Methods of treating and preventing gastrointestinal motility disorders using aminosterols

ABSTRACT

This invention relates to methods of stimulating the activity of the human and animal enteric nervous system. The method comprises orally administering an aminoserol, such as squalamine, a naturally occurring aminosterol isolated from Squalus acanthias, or derivatives thereof, to a subject in need.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/604,452, filed May 24, 2017, which is a divisional application ofU.S. patent application Ser. No. 14/329,627, filed on Jul. 11, 2014,which claims priority to U.S. Provisional Patent Application No.62/015,657, filed on Jun. 23, 2014, and U.S. Provisional PatentApplication No. 61/886,512, filed on Oct. 3, 2013. The contents of theseapplications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to methods of stimulating the activity of thehuman and animal enteric nervous system, which is useful in thetreatment of various diseases or conditions. The method comprises orallyadministering to a subject in need squalamine, a naturally occurringaminosterol isolated from Squalus acanthias, or derivatives thereof. Themethod results in the controlled activation of the intestinal entericnervous system. The method is useful for the treatment ofgastro-intestinal motility disorders such as chronic idiopathicconstipation, Opioid induced constipation, irritable bowel syndrome andinflammatory bowel disease, diabetes, and neurodegenerative diseases,such as Parkinson's disease, Alzheimer's disease, dementia of aging,Huntington's chorea, neuropathy of diabetes, peripheral sensoryneuropathy, traumatic head and/or spine injury, stroke, Amyotrophiclateral sclerosis, multiple sclerosis, depression, epilepsy and autism.In addition, the method is useful for the treatment and prevention of avariety of malignancies, including those of the colon, pancreas, liver,brain, male and female genitourinary tract, lymphatic and blood tissues,lungs, skin, breast, and endometrium.

BACKGROUND OF THE INVENTION

Chemically squalamine presented a structure never before seen in nature,that being a bile acid coupled to a polyamine (spermidine):

The discovery of squalamine, the structure of which is shown above, wasreported by Michael Zasloff in 1993 (U.S. Pat. No. 5,192,756).Squalamine was discovered in various tissues of the dogfish shark(Squalus acanthias) in a search for antibacterial agents. The mostabundant source of squalamine is in the livers of Squalus acanthias,although it is found in other sources, such as lampreys (Yun et al.,“Identification of Squalamine in the Plasma Membrane of White BloodCells in the Sea Lamprey,” Petromyzon marinus,” J. Lipid Res., 48(12):2579-2586 (2007)).

Numerous studies later demonstrated that squalamine exhibits potentantibacterial activity in vitro (Salmi, Loncle et al. 2008).Subsequently, squalamine was discovered to exhibit antiangiogenicactivity in vitro and upon administration to animals (Sills, Williams etal. 1998; Yin, Gentili et al. 2002). As a consequence, squalamine hasbeen evaluated in disease states known to be associated withpathological neovascularization, such as cancer (Sills, Williams et al.1998; Schiller and Bittner 1999; Bhargava, Marshall et al. 2001;Williams, Weitman et al. 2001; Hao, Hammond et al. 2003; Herbst, Hammondet al. 2003; Sokoloff, Rinker-Schaeffer et al. 2004), and vasculardisorders of the eye, including macular degeneration (US2007/10504A12007), retinopathy of prematurity (Higgins, Sanders et al. 2000;Higgins, Yan et al. 2004; US2007/10504A1 2007), cornealneovascularization (Genaidy, Kazi et al. 2002) and diabetic retinopathy(US2007/10504A1 2007).

The utility of squalamine as an anti-infective has been demonstrated invitro against bacteria and fungi (Moore, Wehrli et al. 1993; Rao,Shinnar et al. 2000; Salmi, Loncle et al. 2008). Squalamine is acationic amphipathic substance exhibiting an affinity for membranescomposed of anionic phospholipids (Selinsky, Zhou et al. 1998; Selinsky,Smith et al. 2000). Like other such agents, including magainin andcationic antimicrobial peptides, squalamine is believed to exertantimicrobial action by interacting electrostatically with the membranesof target microorganisms, which generally display anionic phospholipidson the membrane surface exposed to the environment, subsequentlydisturbing their functional integrity, and causing death of the targetedmicrobe (Sills, Williams et al. 1998; Zasloff 2002; Salmi, Loncle et al.2008).

Recent studies have highlighted the efficacy of systemicallyadministered squalamine to prevent or treat viral infections in animals(Zasloff et al., “Squalamine as a broad-spectrum systemic antiviralagent with therapeutic potential,” Proc. Natl. Acad. Sci. USA, 108(38):15978-83 (2011); U.S. (2011) Ser. No. 12/913,648).

The mechanism of action. It has been reported that squalamine exerts itseffects at the cellular level by displacing proteins boundelectrostatically to negatively charged membranes, causing pleiotropicchanges in the functional state of the cell (Alexander et al., “Membranesurface charge dictates the structure and function of the epithelialna+/h+ exchanger,” EMBO J., 30:679-691. (2011); Yeung et al., “Membranephosphatidylserine regulates surface charge and protein localization,”Science, 319(5860): 210-3 (2008); Sumioka et al., “TARP phosphorylationregulates synaptic AMPA receptors through lipid bilayers,” Neuron,66(5): 755-67 (2009); Zasloff et al., “Squalamine as a broad-spectrumsystemic antiviral agent with therapeutic potential,” Proc. Natl. Acad.Sci. USA, 108(38): 15978-83 (2011)).

Aminosterol 1436 is an aminosterol isolated from the dogfish shark,which is structurally related to squalamine (U.S. Pat. No. 5,840,936;Rao, Shinnar et al. 2000). Aminosterol 1436 exhibits antiviral activityagainst HIV in tissue culture (U.S. Pat. No. 5,763,430) via a mechanismproposed to involve inhibition of a lymphocyte-specific NHE by 1436,resulting in suppression of cytokine responsiveness, and subsequentdepression of the capacity of the lymphocyte to support HIV replication(U.S. Pat. No. 5,763,430). Aminosterol 1436, however, has an additionalpharmacological property, not shared with squalamine, namely potentappetite suppression and promotion of dose-dependent weight loss (U.S.Pat. No. 6,143,738; Ahima et al., “Appetite suppression and weightreduction by a centrally active aminosterol.” Diabetes, 51(7): 2099-104(2002); Patel et al., 2002).

Prior clinical studies in humans have focused on the anti-angiogenicproperties of squalamine. Squalamine in its intravenous form, squalaminelactate, is in the process of being tested as a treatment forfibrodysplasia ossificans progressiva, a rare disease where connectivetissue will ossify when damaged. Genesis, A., “Squalamine trial for thetreatment of fibrodysplasia ossificans progressiva initiated”,Angiogenesis Weekly, 8:45 (2002). Squalamine is also undergoing trialsfor treatment of non-small cell lung cancer (stage IIIA) as well asgeneral phase I pharmacokinetic studies. Herbst et al., “A Phase IIIATrial of Continuous Five-Day Infusion of Squalamine Lactate (MSI-1256F)Plus Carboplatin and Paclitaxel in Patients with Advanced Non-Small CellLung Cancer 1,” Clinical Cancer Research, 9:4108-4115 (2003); Hao etal., “A Phase I and Pharmacokinetic Study of Squalamine, an AminosterolAngiogenesis Inhibitor”, Clin Cancer Res., 9(7): 2465-2471 (2003). In2005, the Food and Drug Administration granted squalamine Fast Trackstatus for approval for treatment of age-related macular degeneration.CATE: California Assistive Technology Exchange,” California AssistiveTechnology Exchange. In 2011, Ohr Pharmaceuticals initiated clinicaltrials to evaluate squalamine lactate, administered as an eye drop, forthe treatment of wet macular degeneration, based on their assessmentthat sufficiently high concentrations of squalamine can access theretina, when the substance is placed onto the corneal surface. Thesestudies are ongoing. Genaera Corporation discontinued trials for the useof squalamine in treating cancer in 2007. “PROSTATE CANCER; GenaeraDiscontinues LOMUCIN in Cystic Fibrosis and Squalamine in ProstateCancer Studies,” Drug Week, pp. 251. 2007-07-20; “Reports describe themost recent news from Genaera Corporation,” Biotech Business Week, pp.1540 (2007 Sep. 17). Squalamine is also marketed under the brand nameSqualamax™ as a dietary supplement, though it has not been approved as adrug in this form and thus cannot make therapeutic claims. Squalamax™ isan unfractionated extract of shark liver, containing innumerableuncharacterized substances in addition to squalamine, and squalamine ispresent in Squalamax™ at less than 0.01% of the total weight of theextract. “Cyber Warning Letter”, Center for Drug Evaluation and Research(2002 May 6). Moreover, the dietary supplement form of squalamine is notpharmaceutical grade squalamine, as pharmaceutical grade squalaminerequires significantly greater manufacturing efforts.

By 2006, over 300 patients had received squalamine in doses ranging from6-700 mg/m²/day by iv administration, in three Phase I and nine Phase IIstudies. Hao et al., “A Phase I and pharmacokinetic study of squalamine,an aminosterol angiogenesis inhibitor,” Clin. Cancer Res., 9:2465-71(2003); Herbst et al., “A phase I/IIA trial of continuous five-dayinfusion of squalamine lactate (MSI-1256F) plus carboplatin andpaclitaxel in patients with advanced non-small cell lung cancer,” Clin.Cancer Res., 9:4108-15 (2003); Bhargava et al., “A phase I andpharmacokinetic study of squalamine, a novel antiangiogenic agent, inpatients with advanced cancers,” Clin. Cancer Res., 7:3912-9 (2001); andConnolly et al., “Squalamine lactate for exudative age-related maculardegeneration,” Ophthalmol. Clin. North Am., 19:381-91 (2006). Thestudies showed that the compound exhibited an acceptable safety profileand evidence of efficacy in these early trials. In 2006 development ofsqualamine was halted for economic/strategic reasons by Genaera. In 2011Ohr Pharmaceuticals initiated studies of the compound administered as aneye drop for the treatment of retinal eye disease, but all studies ofthis compound against cancer have remained in a dormant stage since.

Of relevance to the invention disclosed herein, squalamine has neverbeen studied as an oral agent in a human, and thus its pharmacology andbiological effects in man (and other mammals) are known only afterintravenous administration. Extensive studies in animals have shown thatneither squalamine nor Aminosterol 1436 can be absorbed to any extentfrom the gastrointestinal tract, requiring parenteral administration forthe various previously conceived applications of these compounds.Aminosterol 1436, although capable of inducing weight loss whenadministered parenterally to dogs, and rodents exhibited no anorecticactivity when administered orally, consistent with its poorbioavailability when delivered orally. Indeed, in a published review onthe applications of squalamine as a therapeutic, Genaera scientistsstate “Although squalamine lactate is well absorbed in rodents by thesubcutaneous and intraperitoneal routes, preliminary studies indicatethat it is poorly bioavailable orally.” (Connolly et al., “Squalaminelactate for exudative age-related macular degeneration,” Ophthalmol.Clin. North Am., 19:381-91, (2006)) To date, no published patentapplication or literature reference has documented or reported apharmacological effect of orally administered squalamine (or any otherrelated aminosterol) in humans or animals. (U.S. Pat. Nos. 5,192,756;5,637,691; 5,721,226; 5,733,899; 5,763,430; 5,792,635; 5,795,885;5,840,740; 5,840,936; 5,847,172; 5,856,535; 5,874,597; 5,994,336;6,017,906; 6,143,738; 6,147,060; 6,388,108; 6,596,712; U.S. PatentPublication No. 2005/0261508A1 2005; U.S. Pat. No. 6,962,909; U.S.Patent Publication No. 2006/0166950A1 2006; U.S. Patent Publication No.2006/0183928A1 2006; U.S. Patent Publication No. 2007/10504A1 2007.)

Squalamine and related aminosterols, such as 1436, do not exit thegastrointestinal tract into either the portal or systemic blood stream.This resulted in generally accepted conclusions by those skilled in theart of drug development, as of the year 2014, about 20 years after thereported discovery of squalamine, that squalamine could provide nobenefit for systemic conditions, including malignancies, whenadministered orally.

There remains a need in the art for new method of treating diseases andconditions correlated with stimulation of the activity of the human andanimal enteric nervous system. The present invention satisfies thisneed.

SUMMARY OF THE INVENTION

The present invention is directed to methods of stimulating thegastrointestinal tract to achieve certain medical benefits, as describedherein. The method comprises orally administering a pharmaceuticalcomposition comprising one or more aminosterols to a subject in need. An“aminosterol” can be squalamine or a derivative thereof, Aminosterol1436 or a derivative thereof, or a naturally occurring aminosterolisolated from Squalus acanthias or a derivative thereof, collectivelyreferred to as “squalamine” herein.” The pharmaceutical composition cancomprise one or more pharmaceutically acceptable carriers. The subjectcan be a mammal, including a human.

The invention is based on the discovery of unexpected and unprecedentedactivity of orally administered squalamine and related aminosterols(e.g., Aminosterol 1436). The activity relates to stimulating a sequenceof events within the human GI tract with therapeutic value. The sequenceof events stimulated by an aminosterol such as squalamine or aderivative thereof involves the induction of an intestinal secretoryresponse followed by a period of “small intestinal quieting,” and thesubsequent passage of a normally formed bowel movement. These events arebest explained as a consequence of the stimulation of a heretoforeunknown physiological gastrointestinal response, in this invention shownto be controlled or initiated by an effective oral dose of anaminosterol such as squalamine or the related aminosterol, 1436(Aminosterol-Induced GI Response).

Based on the pharmacology of the response, and the likely knowncomponents of the gastrointestinal tract that have been engaged, it ispossible to predict uses or applications of the methods of theinvention. These uses include: (1) treatment and prevention of disordersof gastrointestinal motility, such as chronic idiopathic constipation,Opioid induced constipation, irritable bowel syndrome, and inflammatorybowel disease; (2) treatment and prevention of conditions such asdiabetes mellitus and diabetic neuropathy; (3) treatment and preventionof disorders of the nervous system that could benefit fromneuro-protection, such as Parkinson's Disease, Alzheimer's disease,Huntington's Disease, acute traumatic injury to the central nervoussystem, including the spinal cord, stroke, acute head and/or spineinjury, degenerative processes associated with aging, including memoryloss (“dementia of aging”), cerebral palsy, epilepsy, peripheral sensoryneuropathy, and multiple sclerosis; (4) treatment or prevention of avariety of malignancies, and particularly vascularized malignancies,including but not limited to malignancies of the colon, pancreas, liver,brain, male and female genitourinary tract, lymphatic and blood tissues,lungs, skin, breast, and endometrium (unexpected responses, as describedherein, include regression of malignancies); (5) treatment or preventionof depression, and (6) treatment or prevention of autism.

The invention comprises orally administering a therapeutically effectiveamount of squalamine or a derivative thereof, an isomer or prodrug ofsqualamine, or a pharmaceutically equivalent salt thereof to a subject,such as a mammal, in need. A “subject in need” is a human or mammal witha disorder in which the stimulation of the “Aminosterol-Induced GIResponse” would provide therapeutic or medical benefit.

Preferably, the squalamine is a pharmaceutical grade squalamine. Thecomposition can further comprise one or more pharmaceutically acceptableexcipients. The squalamine or derivative thereof is present in an amountsufficient to produce the intended benefit or response.

In another embodiment, the invention encompasses methods of treatingand/or preventing conditions benefitted by the stimulation of theAminosterol-Induced GI Response comprising administering atherapeutically effective amount of an aminosterol that can inhibit theformation of actin stress fibers in endothelial cells stimulated by aligand known to induce stress fiber formation. An exemplary aminosteroluseful in the methods of the invention has the chemical structure ofFormula I:

wherein,

-   W is 24S—OSO₃H or 24R—OSO₃H;-   X is 3β-H₂N—(CH₂)₄—NH—(CH₂)₃—NH— or 3α-H₂N—(CH₂)₄—NH—(CH₂)₃—NH—;-   Y is 20R—CH₃; and-   Z is 7α or 7β-OH.

In another embodiment of the invention, the aminosterol is one of thenaturally occurring aminosterols (1-8) isolated from Squalus acanthias:

In one embodiment, the aminosterol is Aminosterol 1436 or a squalamineisomer.

In yet another embodiment of the invention, the aminosterol is aderivative of squalamine or another naturally occurring aminosterolmodified through medical chemistry to improve biodistribution, ease ofadministration, metabolic stability, or any combination thereof. Inanother embodiment, the squalamine or aminosterol is modified to includeone or more of the following: (1) substitutions of the sulfate by asulfonate, phosphate, carboxylate, or other anionic moiety chosen tocircumvent metabolic removal of the sulfate moiety and oxidation of thecholesterol side chain; (2) replacement of a hydroxyl group by anon-metabolizable polar substituent, such as a fluorine atom, to preventits metabolic oxidation or conjugation; and (3) substitution of variousring hydrogen atoms to prevent oxidative or reductive metabolism of thesteroid ring system.

In yet another embodiment, the aminosterol comprises a sterol nucleusand a polyamine, attached at any position on the sterol, such that themolecule exhibits a net charge of at least +1, the charge beingcontributed by the polyamine.

In yet another embodiment, the aminosterol comprises a bile acid nucleusand a polyamine, attached at any position on the bile acid, such thatthe molecule exhibits a net positive charge being contributed by thepolyamine.

In certain embodiments of the invention, the methods compriseadministering squalamine or a derivative thereof at an effective dailydosing amount of about 0.1 to about 20 mg/kg body weight. In certainembodiments, the effective dose can be established by defining theinitial dose required to induce the Aminosterol-Induced GI Response,i.e., the initial dose required to stimulate nausea and secretorydiarrhea.

The composition can be administered via any pharmaceutically acceptablemethod, including but not limited to oral administration.

The methods of the invention can further comprise administering thesqualamine or derivative thereof in combination with at least oneadditional active agent to achieve either an additive or synergisticeffect. Such an additional agent can administered via a method selectedfrom the group consisting of concomitantly, as an admixture, separatelyand simultaneously or concurrently, and separately and sequentially.

In one embodiment of the invention, the oral dosage form is a liquid,capsule, or tablet designed to disintegrate in either the stomach, uppersmall intestine, or more distal portions of the intestine with adissolution rate appropriate to achieve the intended therapeuticbenefit.

In another embodiment of the invention, essentially no aminosterol isdetected in the blood stream of the subject following oraladministration.

Both the foregoing summary of the invention and the following detaileddescription of the invention are exemplary and explanatory and areintended to provide further details of the invention as claimed. Otherobjects, advantages, and novel features will be readily apparent tothose skilled in the art from the following detailed description of theinvention.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1C shows that intraluminal squalamine increases mesentericnerve firing frequency. FIG. 1A shows a representative trace of asuction electrode multiunit recording. The insert in FIG. 1A showsextracellular action potential (40 μV) on a faster timebase (1 ms). FIG.1B shows a histogram of multiunit firing frequency averaged over 1 minbins. Squalamine was applied into the lumen by gravity feed 11 min afterbeginning the recording. FIG. 1C shows the before and after data ofspike frequencies averaged over 3 min for 5 separate experiments. TheControl represents background discharge before applying squalamine, andthe Peak gives the average firing frequency during a 3 min period at thepeak of the response.

FIG. 2A shows that intraluminal application of squalamine had noapparent effect on mouse colon migrating motor complex (MMC) peakpressure waves, although FIG. 2B shows that MMC propagation velocityfrom oral to anal was increased.

FIG. 3 shows a representative recording of IPAN member potential.Squalamine evoked bursts of action potential lasting for 10 to 30 minafter application of a brief (20 ms) 50 μL puff of squalamine onto theepithelium.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods of stimulating astereotyped pharmacological response in the human gastro-intestinaltract following oral administration of squalamine or a derivativethereof, or Aminosterol 1436 or a derivative thereof (AminosterolInduced GI Response). The invention is unexpected and surprising basedon the known and predicted properties of aminosterols, includingsqualamine and Aminosterol 1436. In particular, the invention permitsexerting pharmacologic control over the enteric nervous system in amanner that is without precedent in the literature. The utility affordedby this capability includes all applications in which activation of theenteric system in this fashion could have benefit. These applicationsinclude GI conditions that would benefit from the imposition of a periodof small intestinal “quieting,” resembling what is commonly called an“ileal brake,” or from direct effects on the enteric nervous system ofamino-sterol specific activation imposed by Aminosterol administration.

An example of a condition that can be treated with a method according tothe invention includes diabetes, where the delayed transit of foodthrough the small intestine would reduce the rate of nutrient absorptionand secondarily reduce stress on the endocrine pancreas. Otherconditions that can be treated using a method according to the inventioninclude irritable bowel syndrome, Opioid-induced constipation, andinflammatory bowel disease, where relaxation of the smooth muscle of thesmall intestine would provide relief of cramping peristaltic activity.Yet other conditions that can be treated with a method according to theinvention include neurodegenerative diseases which would benefit fromthe direct effects of the aminosterols on the enteric neurons and theircommunication with immune cells within the lamina propria, as well asthe stimulation of vagal afferents that track to higher centers of thecentral nervous system, such as Parkinson's disease, Alzheimer'sdisease, Huntington's chorea, neuropathy of diabetes, peripheral sensoryneuropathy, traumatic head and/or spine injury, stroke, Amyotrophiclateral sclerosis, multiple sclerosis, depression, epilepsy and autism.Finally, methods according to the invention are also useful in treatingand preventing a variety of malignancies, including for example, anyvascularized malignancy, such as a malignancy of the colon, pancreas,liver, brain, male and female genitourinary tract, lymphatic and bloodtissues, lungs, skin, breast, and endometrium. Unanticipated benefitsinclude regression of malignancies.

I. Definitions

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

As used herein the term “aminosterol” encompasses squalamine or aderivative thereof, an isomer or prodrug of squalamine, Aminosterol 1436or a derivative thereof, an isomer or prodrug of Aminosterol 1436, or anaturally occurring aminosterol isolated from Squalus acanthias or aderivative thereof, as described herein. “Aminosterols” useful in theinvention also encompass a pharmaceutically equivalent salt of anyaminosterol compound described herein. These compounds, andpharmaceutically acceptable salts thereof, are collectively referred toherein as “squalamine” and “aminosterols.” Thus, the term “aminosterol”as used herein is intended to encompass the broader class that includesboth squalamine and the known naturally occurring aminosterols.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term.

As used herein, “therapeutic activity” or “activity” may refer to anactivity whose effect is consistent with a desirable therapeutic outcomein humans, or to desired effects in non-human mammals or in otherspecies or organisms. Therapeutic activity may be measured in vivo or invitro. For example, a desirable effect may be assayed in cell culture.

As used herein, the phrase “therapeutically effective amount” shall meanthe drug dosage that provides the specific pharmacological response forwhich the drug is administered in a significant number of subjects inneed of such treatment. It is emphasized that a therapeuticallyeffective amount of a drug that is administered to a particular subjectin a particular instance will not always be effective in treating theconditions/diseases described herein, even though such dosage is deemedto be a therapeutically effective amount by those of skill in the art.

II. Mechanism of Action

The mechanism of action. It has been reported that squalamine exerts itseffects at the cellular level by displacing proteins boundelectrostatically to negatively charged membranes, causing pleiotropicchanges in the functional state of the cell. See Alexander et al.,“Membrane surface charge dictates the structure and function of theepithelial na+/h+ exchanger,” EMBO J., 30:679-691. (2011); Yeung et al.,“Membrane phosphatidylserine regulates surface charge and proteinlocalization,” Science, 319(5860): 210-3 (2008); Sumioka et al., “TARPphosphorylation regulates synaptic AMPA receptors through lipidbilayers,” Neuron, 66(5): 755-67 (2009); and Zasloff et al., “Squalamineas a broad-spectrum systemic antiviral agent with therapeuticpotential,” Proc. Natl. Acad. Sci. USA, 108(38): 15978-83 (2011). Withrespect to the disclosed invention, it is believed that squalamine andother aminosterols, such as Aminosterol 1436, are transported into theintestinal enterocyte. The presence of the aminosterol induces aresponse within the enterocyte, including effects on water and saltreabsorption. The aminosterol is then transported into the laminapropria where it then enters certain neurons of the enteric nervoussystem (via specific transporters) and induces electrical activation,ultimately, by the electrostatic mechanism proposed. The bulk of theaminosterol is then likely pumped back into the intestinal lumen,wherein it is excreted in the feces.

Squalamine is known to gain access to nerve cells, neutralize thenegative electrostatic surface potential of these cells, and alterelectrical channel activity (Sumioka et al., 2009). It is assumed thatsqualamine can access and influence the behavior of the neurons of theenteric nervous system in a fashion similar to what has been observed incortical granular neurons (Sumioka et al., 2009). In addition,squalamine is known to inhibit the sodium hydrogen exchanger involved inwater and salt reabsorption in the human small intestine by the samemechanism (Alexander et al. 2011).

As described in Example 3 below, optimal oral dosing appears to be on anempty stomach. Squalamine, because of its physical properties, isexpected to bind tightly to foodstuff, and be unavailable to interactwith the intestinal epithelium. Only as the food material is digested issqualamine freed. Such would be occurring in the more distal intestine.

Based on the stereotyped nature of the response and known properties ofcertain human gastrointestinal hormones and the communication known toexist between the human GI tract and the central nervous system, thephysiological events that underlie the Aminosterol Induced GI Responsecan be detailed. The observed response can be divided into 3 phases.

Phase I: Nausea. This phase begins within about 1 to about 3 hoursfollowing oral ingestion of an aminosterol, and lasts about 30 minutes.Phases II and III (see below) can be induced at doses below thatrequired to stimulate a conscious sensation of nausea, so the consciousexperience is not a required component of the overall AminosterolInduced GI Response. The dose required to induce nausea is greater thanthat required to initiate Phases II and III. It is proposed that thesensation of nausea after administration of squalamine or aminosterol1436 is a consequence of the direct stimulation of the brainstem viavagal afferents stimulated within the intestinal wall. The stereotypednature of the response and the predictability of its temporal duration,suggest that the aminosterol stimulates a specific set of entericneurons via a specific mechanism. Were the nausea an effect secondary tonon-specific mucosal injury, it would be expected to be more variable inboth intensity, onset of appearance, and duration.

Based on the timing of the nausea, the site of action is likely theproximal small intestine, the duodenum, and/or the jejunum. It ispossible that nausea experienced at higher doses of aminosterols resultsfrom the discharge of intestinal Enterochromaffin cells, which releasehistamine and serotonin, the L-cells (GLP-1), the K-cells (GIP), and theI-cells (CCK), each releasing hormones that are known to circulatesystemically, exhibit a brief lifetime in the blood stream, and causenausea.

In Example 6 it is shown that orally administered squalamine does notinduce release of GLP-1 into the blood stream of a healthy human, andthus it is likely that Phase I results from vagal afferent stimulationof the nausea centers in the brain. This explanation is supported byExample 10 (FIG. 1), where it is shown that application of squalamine tothe mouse colon leads to the stimulation of electrical signals flowingthrough the afferent arm of the vagus.

Phase II: Net fluid loss from the intestine: If a sufficiently largedose of aminosterol is administered orally, the subject will experiencethe discharge rectally of a small volume of watery fluid. The dischargeis clear, watery in nature and reflects either an increased secretoryresponse or decreased absorptive response of the intestine with respectto its handling of the fluxes of fluid within the lumen. VIP (vasoactiveintestinal peptide) is a well characterized neuropeptide, present withinthe enteric nervous system and well known to provoke this type ofsecretory response. It is presumed that the discharge of the entericnervous system in Phase I triggers the activation of VIP expressingneurons within the enteric nervous system, resulting in the alterationof fluid handling by the enterocytes within the jejunum. Alternatively,the aminosterol could inhibit the sodium-hydrogen exchanger type III(NHE-3) expressed on the lumenal surface of the enterocytes, which isthe major transporter responsible for the absorption of sodium and waterfrom the intestine.

It has previously been reported that squalamine inhibits the NHE-3transporter by an electrostatic mechanism, where squalamine enters anepithelial cell via a specific transporter (Alexander et al. 2011), andthis is the same mechanism described above with respect to the claimedmethod. To access the enteric nervous system, the aminosterol must firstcross the epithelial layer that separates the lumen of the intestinefrom the wall of the bowel, where the neurons of the enteric nervoussystem are situated. It is presumed that squalamine, and other activeaminosterols, cross the epithelium principally through the transportinto the epithelial cell, followed by the subsequent exiting of themolecule from that cell. During the period of time squalamine remains inthe cell (and likely for some time after), it is expected that the NHE-3transporter is inhibited, and effects on fluid flux within that segmentof intestine accordingly affected. The duration of the response and thereproducible volume released suggests that the response isself-limiting, possibly through a negative/inhibitory feedback loopinvolving the eneteric nervous system, or as a consequence of theclearance from the epithelial cell of the compound and the subsequentrestoration of normal function. Regardless of the mechanism, thestereotypic and dose-dependent nature of the secretory response suggestthat the administration of the aminosterol stimulates the smallintestine in a highly specific fashion.

Phase III: Intestinal Motility. Squalamine has distinct pharmacologicaleffects on both small and large intestinal motility and muscle tone.Following the watery discharge, the bowel enters a period of “quiet”that lasts between 2-3 days, following the larger squalamine dose (forexample 200 mg, although other larger doses as described herein can beused). At lower squalamine doses, the period of “quiet” is reducedproportionally in duration. During this period the bowel is not inactive(“ileus”) as it would be following oral administration of an opioidnarcotic. There are bowel sounds, and gas is passed intermittently.Appetite is nearly normal, although it could in principal be slightlyreduced. Gastric fullness after a meal is sensed. During this phase, onedoes not experience bloating, abdominal discomfort, abdominal pain,nausea, or a sense of fullness. The delay in the passage of a stool willbe recognized as “unusual” in an individual with a more frequent bowelpattern. This phase ends with passage without urgency of a soft stool,in contrast to what would have been observed following a period of“constipation.” This phase is either a direct effect of squalamine onthe enteric nervous system, progressively stimulating the entericnervous system as the compound moves rectally, and/or a result of theactivation of the brain by the presence of squalamine within the gutresulting in efferent signals directed by the brain that alter themotility of the intestines.

This Phase is reminiscent of the condition of reduced gut motilitytermed an “ileal brake.” It is designed physiologically to slow gutmotility to enhance nutrient extraction. Certain gut hormones, such asGLP-1, exhibit this pharmacological activity. Indeed, the benefits ofGLP-1 and its analogs in the treatment of diabetes mellitus in largepart are believed to derive from the ability of GLP-1 and analogsthereof to slow entry of nutrients from the intestine into the liver,and thereby reduce the secretory rate of insulin required to match theinflux of nutrients. However, as shown in Example 6, oral administrationof squalamine does not stimulate release of GLP-1 as measured in theblood stream. It can also be explained as a consequence of theactivation of VIP-nergic neurons within the enteric nervous. The releaseof VIP from these neurons is directed at the muscle layers of the bowelwall and has a known relaxing effect on muscular contractions of theintestine. It is believed that this mechanism is the most plausible.This mechanism could arise via direct stimulation of these neurons bythe presence of squalamine, or indirectly, via the stimulation ofspecific signals from the enteric nervous system directed to the brain,which in turn sends out signals that release VIP from enteric nerves.

Since squalamine is not absorbed from the GI tract, it moves slowlytoward the rectum following oral ingestion. As squalamine proceedsdistally within the small intestine, squalamine stimulates theunderlying epithelium (increasing the fluid content of the lumen) andstimulates the underlying enteric nervous system. Over the course ofabout 2 to about 3 days, the movement of squalamine into the colon andeventually its excretion in feces leads to the termination of itspharmacological activity and restoration of prior bowel function.

The effects of squalamine on the colon appear to differ from those onthe small intestine. In particular, it appears that squalamine increasescolonic motility as part of the Aminosterol-induced GI response, basedon the soft nature of the stool produced in a treated human. Inaddition, studies in the mouse (Example 11, FIG. 2) confirm thatsqualamine stimulates colonic motility. Thus, while small intestinalmotility appears to be slowed, colonic motility appears to be increased,with a compensatory adjustment of net fluid flux within the bowel tomaintain a stool of normal consistency.

III. Beneficial Consequences of the Aminosterol-Induced GI Response

Based on the unanticipated pharmacological response of the GI tract tooral administration of squalamine, several unexpected and unprecedentedapplications can be understood.

Chronic Idiopathic Constipation, Opioid-induced constipation, IrritableBowel Syndrome and Inflammatory Bowel Disease: The fundamental etiologyof these common conditions is not known. Two broad categories ofIrritable Bowel Syndrome exist, one characterized by diarrhea, the otherby constipation. Amongst the more effective of the treatments areserotonin analogs that act on the enteric nervous system to eitherstimulate mobility (in the constipation form) or inhibit it (in thediarrheal form). Treatment of IBS with an oral aminosterol could“reboot” the enteric nervous system. Clearly, in the case of thediarrheal form, administration should impose an about 2 to about 5 dayperiod of bowel rest/silence that would be expected to reduce smallintestinal transit. In the case of the constipation type, the impositionof a muscle-relaxing effect on the small intestine and a stimulation ofcolonic mobility of the GI tract could introduce normalcy. Thestimulation of net intestinal secretion along with imposition of the“controlled” motility patterns of the small and large intestines wouldprovide benefit in the setting of chronic constipation. Similarly, amethod according to the invention can provide benefit in the setting ofopioid-induced constipation, where secretion is inhibited, peristalticcontractions become uncoordinated, and colonic motility is markedlyreduced, as oral aminosterol administration should address these issues.

Diabetes mellitus: The use of GLP-1 analogs in the treatment of diabeteshas been well established. These compounds reduce the insulinrequirement and tend to smooth out overall insulin titration. Themechanism by which the GLP-1 compound class produces their benefitremains controversial, with some arguments presented in support ofpromoting insulin secretion, while other arguments support thebeneficial effects of the “ileal brake.” Thus, the method according tothe invention utilizing oral administration of one or more aminosterols,to trigger the Aminosterol-Induced GI Response, should prove beneficialalong with insulin/and insulin secretagogues in the management ofDiabetes mellitus.

Neurodegenerative disorders: The proposed mechanism by which squalamineprovokes the Aminosterol Induced GI Response involves the directstimulation of nerves within the enteric nervous system, and stimulationof currents flowing towards the brain through afferent nerves of thevagus. Stimulation of afferents of the vagus, which distribute tocenters and tracts within the brain would be expected to stimulaterelease of a suite of neuropeptides within the brain itself. Thecontinued imposition of the ileal brake for several days followingaminosterol dosing, speaks to the length of time theaminosterol-provoked gut/CNS interaction must be operative following asingle dose of squalamine.

In addition, the entry of squalamine into the nerves of the entericnervous system could provide direct benefit in degenerative conditionswhere accumulation of certain proteins is believed to be causallyinvolved. Specifically, in Parkinson's disease, the accumulation ofalpha synuclein is believed to play a role in the neuronal damageassociated with the condition. Alpha synuclein is a protein with acationic N-terminus and can interact electrostatically with the internalmembranes of the nerve cell in which it is expressed. Since squalaminecan both enter nerve cells and neutralize the negative surface potentialof these membrane surfaces, squalamine and related aminosterols have thecapacity to displace alpha synuclein from membrane sites within nerves,and as a consequence, interrupt the pathophysiology of the disease. Thisprinciple is demonstrated in Example 14.

Cancer Therapy: The complex interactions between the epithelial cellsand enteric neurons suggest that the methods of the invention caninfluence the growth and spread of cancer. Recent studies in bothanimals and man have strongly suggested that malignant tumors mustestablish communication with the autonomic nervous system. The enhancedflux of electrical signals emanating from the squalamine-stimulatedenteric nervous system could disrupt effective communication between amalignant tumor and the divisions of the autonomic nervous system.Indeed, as shown in Examples 14-19, the induction of the AminosterolInduced GI Response is associated with striking regression of currentlyuntreatable malignancies, under conditions where squalamine itself doesnot enter the bloodstream, and therefore the aminosterol must be actingin an indirect fashion. Examples of malignancies that can be treatedusing the methods of the invention include, but are not limited to,vascularized malignancies, and/or malignancies of the colon, pancreas,liver, brain, male and female genitourinary tract, lymphatic and bloodtissues, lungs, skin, breast, and endometrium. All of these cancers areknown to be influenced by metastatic spread through the lymphatic andvascular systems, a process which we believe to be influenced by theaction of the autonomic nervous system.

Thus, the invention disclosed herein teaches how to stimulate theenteric nervous system to achieve certain beneficial effects in manydifferent diseases and leads to the possibility that by administering anaminosterol such as squalamine or a derivative thereof under conditionsthat provoke the Aminosterol-Induced GI Response, neuroprotectivebenefits can accrue to an individual so treated.

The conditions referred to are those in which the induction ofneuro-protective hormones could provide preventative or therapeuticbenefit. These conditions include, for example, Parkinson's disease,Alzheimer's disease, Stroke, Amyotrophic lateral sclerosis, Acutetraumatic injury to the central nervous system, including the spinalcord, neurodegenerative processes of aging, early stages of cerebralpalsy, epilepsy, peripheral sensory neuropathy, diabetic neuropathy,Huntington's chorea, Multiple sclerosis, depression and autism. Inaddition, by administering squalamine under conditions that provoke theAminosterol-Induced GI Response, certain human malignancies can beinduced to regress, in a setting where orally administered squalaminehas not entered the bloodstream.

Experiments in animal models of neurodegenerative disease havedemonstrated the neuro-protective benefits of several of theneuropeptides likely to be released within the nervous system during theunfolding Aminosterol-Induced GI Response. These include:

-   -   Vasoactive intestinal peptide: Alzheimer's (White et al. 2010);        Parkinson's (Delgado and Ganea, 2003); Head trauma (Gressens,        Marret et al, 1997); multiple sclerosis (Gonzalez-Rey,        Fernandez-Martin et al. 2006)    -   GLP-1: Parkinson's (Li, Perry et al. 2009); head trauma (Li,        Perry et al. 2009); Alzheimer's (Li, Perry et al. 2009).    -   CCK: Epilepsy (Tirassa, Costa et al. 2005)

Clinical studies in man have demonstrated the benefits of vagal nervestimulation in several disorders, and as a consequence of vagal afferentactivity induced by the Aminoterol-Induced GI Response, severalconditions can be considered for which oral aminosterol administrationcould provide clinical benefit, such as depression, epilepsy and autism.

IV. Beneficial Pharmacological Properties of the Aminosterols asStimulants of the Aminosterol-Induced GI Response

Aminosterols, such as squalamine and derivatives thereof, including butnot limited to Aminosterol 1436, are not absorbed from thegastrointestinal tract of mammals, including man. As a consequence, thisinvention teaches how to stimulate the Aminosterol-Induced GI Responsewithout introducing aminosterols into the human systemic circulation.This is significant, as toxicities known to be associated with, forexample, injectable administration of Aminosterols and derivativesthereof, are not a concern for the uses disclosed in this invention. Inaddition, issues relating to potential toxicities not as yet known(effects on fertility, etc) would be of lesser concern with the oraladministration protocol of the invention.

In one embodiment of the invention, following oral administration thereis essentially no detectable levels of the administered aminosterol inthe bloodstream of the subject. In another embodiment of the invention,following oral administration there is preferably less than about 10ng/ml of the administered aminosterol in the bloodstream of the subject,measured between about 1-about 12 hours following oral administration.

Retention of the aminosterols within the lumen of the intestine permitsoptimal orchestration of the Aminosterol-Induced GI Response. As theeffects on intestinal motility that characterize Phase III are actuated,and gut motility within the small intestine is slowed, the passagedistally of the aminosterol is slowed. Thus, the intensity of theAminosterol-Induced GI Response will be maintained by “a positivefeedback loop,” whereby the slowing of small intestinal motility extendsthe duration of effect until it reaches the colon, where it is finallyexpelled, resulting in the termination of the Aminosterol-Induced GIResponse.

Repeat dosing regimens are timed by the rate of clearance of theaminosterol from the intestine. It is assumed that at a certain timeafter the initial “loading” dose, surface concentrations of theaminosterol will decrease as the substance spreads across the surface ofthe intestinal walls and progresses distally. In the examples describedbelow, the Aminosterol-Induced GI Response appears to last about 4 daysfollowing a single 200 mg oral dose of squalamine or Aminosterol 1436. Asecond dose on day 4 of about 100 mg, followed by successive about 100mg dosing every 4 days, would represent one reasonable regimen designedto maintain a steady state surface concentration in the intestine.

Effective dosing regimens can also be clinically established based onthe dose required to observe a change in bowel behavior at least about1-about 2 hours following oral dosing. A change in bowel behaviorincludes a change in the normal frequency of defecation, the consistencyof the stools, the perceived activity of the bowels, nausea, or thepassage of a watery rectal discharge. An effective oral dose generallyfalls between about 10 mg to about 400 mg.

Dosing can be once daily, or divided over multiple time periods duringthe day.

Exemplary dosing regimens include, but are not limited to: (1)Initiating with a “low” initial daily dose, and gradually increasing thedaily dose until a dose is reached that elicits evidence of theactivation of the enteric nervous system, where the “low” dose is fromabout 10-about 100 mg per person, and the final effective daily dose isbetween about 25-about 1000 mg/person; (2) Initiating with a “high”initial dose, which necessarily stimulates the enteric nervous system,and reducing the subsequent daily dosing to that required to elicit aclinically acceptable change in bowel behavior, with the “high” dailydose being between about 50-about 1000 mg/person, and the subsequentlower daily oral dose being between about 25-about 500 mg/person; (3)Periodic dosing, where an effective dose can be delivered once everyabout 2, about 3, about 4, about 5, about 6 days, or once weekly, withthe initial dose determined to capable of eliciting an AminosterolInduced Response

Oral dosing should continue at least until the clinical condition hasresolved. To establish the need for continued dosing, treatment can bediscontinued and the condition reevaluated. If necessary, oraladministration should be resumed. The period of oral dosing can be forabout 1, about 2, about 3, or about 4 weeks; about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, or about 12 months, or about 1, about 2, about 3, about 4, or 5years, or longer.

Failure to elicit an Aminosterol-Induced GI Response would suggest thatthe dose being administered was inadequate, and would suggest continuedtitration until the GI response is observed. Dosing could proceed from alower to higher dose, with the GI responses to daily increasesmonitored. An effective dose would be that which induced the complete GIresponse. An excessive Aminosterol-Induced GI Response at a low doseswould speak to a sensitivity and would guide appropriate administrationat lower doses.

The sensitivity of the Aminosterol-Induced GI Response to oraladministration of aminosterols is likely due to several variables: (1)The absorption of the aminosterol into a mucous layer, an effect thatwould reduce free concentration of aminosterol available for diffusiononto the epithelial surface, thereby reducing the response to a givenoral dose; and (2) an increase in the permeability of the epithelialwall (leakiness), which occurs following infections, allergicenteropathies, and in states of intestinal inflammation. In suchsettings, the normal transport of the aminosterol across the epithelium,which is facilitated by the controlled entry and subsequent exit of themolecule from the lining epithelial cell, would be circumvented.Compound would leak across the epithelial barrier, and expose the nervenetwork within the bowel wall to abnormally high concentrations. Hence,an excessive response might provide a diagnostic impression of thepermeability status of the epithelium.

V. Compositions Useful in the Methods of the Invention

The invention relates to methods of treating conditions that benefitfrom stimulation of the Aminosterol-Induced GI Response. The methodscomprise orally administering a therapeutically effective amount of oneor more aminosterols or a pharmaceutically equivalent salt thereof to asubject in need. A “subject in need” is a human or animal at risk of orsuffering from conditions including, but not limited to, Irritable bowelsyndrome, Opioid-induced constipation, Inflammatory Bowel Disease,Diabetes mellitus, Parkinson's disease, Alzheimer's disease, dementia ofaging, Huntington's chorea, neuropathy of diabetes, peripheral sensoryneuropathy, traumatic head and/or spine injury, stroke, Amyotrophiclateral sclerosis, multiple sclerosis, depression, epilepsy and autism.In addition, by orally administering an aminosterol under conditionsthat provoke the Aminosterol-Induced GI Response, certain humanmalignancies can be induced to regress, in a setting where theaminosterol has not entered the bloodstream. Similarly, chronic oraladministration of one or more aminosterols should prevent the appearanceof malignancy.

U.S. Pat. No. 6,962,909, for “Treatment of neovascularization disorderswith squalamine” to Zasloff et al., discloses various aminosterols, thedisclosure of which is specifically incorporated by reference. Anyaminosterol known in the art, including those described in U.S. Pat. No.6,962,909, can be used in this invention, as long as the aminosterolcarries a net positive charge of at least +1 created by a polyaminemoiety.

In yet another embodiment, the aminosterol comprises a bile acid nucleusand a polyamine, attached at any position on the bile acid, such thatthe molecule exhibits a net positive charge being contributed by thepolyamine.

In another embodiment, the invention encompasses methods of treatingconditions described herein comprising orally administering atherapeutically effective amount of one or more aminosterols that caninhibit the formation of actin stress fibers in endothelial cellsstimulated by a ligand known to induce stress fiber formation. Theaminosterols can have the chemical structure of Formula I:

wherein,

-   W is 24S—OSO₃H or 24R—OSO₃H;-   X is 3β-H₂N—(CH₂)₄—NH—(CH₂)₃—NH— or 3α-H₂N—(CH₂)₄—NH—(CH₂)₃—NH—;-   Y is 20R—CH₃; and-   Z is 7α or 7β-OH.

Exemplary aminosterols that can be used in the methods of the inventioninclude, but are not limited to, the known aminosterols (compounds 1-8)isolated from Squalus acanthias:

A variant or derivative of squalamine may have one or more chemicalmodifications which do not modify the activity of squalamine. Similarly,analogous modifications can be made to the other known naturallyoccurring aminosterols described above. A “variant” or “derivative” ofsqualamine or a naturally occurring aminosterol in which modificationswell known in the art of medicinal chemistry to “mimic” the originalspatial and charge characteristics of a portion of the originalstructure have been introduced to improve the therapeuticcharacteristics of the aminosterol. In general, such modifications areintroduced to influence metabolism and biodistribution. Examples of suchvariants or derivatives include, but are not limited to, (1)substitutions of the sulfate by a sulfonate, phosphate, carboxylate, orother anionic moiety chosen to circumvent metabolic removal of thesulfate moiety and oxidation of the cholesterol side chain; (2)replacement of an hydroxyl group by a non-metabolizable polarsubstituent, such as a fluorine atom, to prevent its metabolic oxidationor conjugation; and (3) substitution of various ring hydrogen atoms toprevent oxidative or reductive metabolism of the steroid ring system.

The compositions useful in the methods of the invention comprise atleast one aminosterol. In one embodiment, the compositions used in themethods of the invention comprise: (a) at least one pharmaceutical gradeaminosterol; and optionally (b) at least one phosphate selected from thegroup consisting of an inorganic phosphate, an inorganic pyrophosphate,and an organic phosphate, wherein the aminosterol is formulated as aweakly water soluble salt of the phosphate. In another embodiment of theinvention, the phosphate is an inorganic polyphosphate, and the numberof phosphates can range from 3 (tripolyphosphate) to 400. In yet anotherembodiment, the phosphate is an organic phosphate which comprisesglycerol 2 phosphates. In yet another embodiment, the aminosterol isselected from the group consisting of: (a) squalamine or apharmaceutically acceptable salt or derivative thereof; (b) a squalamineisomer; (c) Aminosterol 1436; (d) an aminosterol comprising a sterol orbile acid nucleus and a polyamine, attached at any position on thesterol or bile acid, such that the molecule exhibits a net charge of atleast +1, the charge being contributed by the polyamine; (e) anaminosterol which is a derivative of squalamine modified through medicalchemistry to improve biodistribution, ease of administration, metabolicstability, or any combination thereof; (f) an aminosterol modified toinclude one or more of the following: (i) substitutions of the sulfateby a sulfonate, phosphate, carboxylate, or other anionic moiety chosento circumvent metabolic removal of the sulfate moiety and oxidation ofthe cholesterol side chain; (ii) replacement of a hydroxyl group by anon-metabolizable polar substituent, such as a fluorine atom, to preventits metabolic oxidation or conjugation; and (iii) substitution ofvarious ring hydrogen atoms to prevent oxidative or reductive metabolismof the steroid ring system; (g) an aminosterol that can inhibit theformation of actin stress fibers in endothelial cells stimulated by aligand known to induce stress fiber formation, having the chemicalstructure of Formula I (above).

In one embodiment, the methods of the invention can employ a formulationof Aminosterol 1436 (Zasloff, Williams et al. 2001) as an insoluble saltof phosphate, polyphosphate, or an organic phosphate ester. In anotherembodiment, the aminosterol can be composed of a sterol or bile acidnucleus to which a polyamine is chemically linked, displaying a netpositive charge of at least +1. The invention can be embodied in aformulation comprising a phosphate suspension or as a tablet for oraladministration. As an oral formulation, squalamine phosphate wouldslowly dissolve in the gastrointestinal tract, and not subject thelining of the intestine to high local concentrations that wouldotherwise irritate or damage the organ.

Dosage Forms. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Any pharmaceutically acceptable dosage form may beemployed in the methods of the invention For example, the compositioncan be formulated into a dosage form (a) selected from the groupconsisting of liquid dispersions, gels, aerosols, lyophilizedformulations, tablets, capsules; and/or (b) into a dosage form selectedfrom the group consisting of controlled release formulations, fast meltformulations, delayed release formulations, extended releaseformulations, pulsatile release formulations, and mixed immediaterelease and controlled release formulations; or (c) any combination of(a) and (b).

An exemplary dosage form is an orally administered dosage form, such asa tablet or capsule. Such methods include the step of bringing intoassociation the aminosterol with the carrier that constitutes one ormore accessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and then,if necessary, shaping the product.

Formulations or compositions of the invention may be packaged togetherwith, or included in a kit with, instructions or a package insert. Forinstance, such instructions or package inserts may address recommendedstorage conditions, such as time, temperature and light, taking intoaccount the shelf-life of the aminosterol. Such instructions or packageinserts may also address the particular advantages of the aminosterol,such as the ease of storage for formulations that may require use in thefield, outside of controlled hospital, clinic or office conditions.

The aminosterol composition can also be included in nutraceuticals. Forinstance, the aminosterol composition may be administered in naturalproducts, including milk or milk product obtained from a transgenicmammal which expresses alpha-fetoprotein fusion protein. Suchcompositions can also include plant or plant products obtained from atransgenic plant which expresses the aminosterol. The aminosterol canalso be provided in powder or tablet form, with or without other knownadditives, carriers, fillers and diluents. Exemplary nutraceuticals aredescribed in Scott Hegenhart, Food Product Design, December 1993.

The aminosterol composition will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the aminosterol alone), the method of administration, thescheduling of administration, and other factors known to practitioners.The “effective amount” for purposes herein is thus determined by suchconsiderations.

Effective dosing regimens can be based on that dose required to observea change in bowel behavior at least about 1-about 2 hours following oraldosing. A change in bowel behavior includes a change in the normalfrequency of defecation, the consistency of the stools, the perceivedactivity of the bowels, nausea, or the passage of a watery rectaldischarge. An effective oral dose generally falls between about 10 mg toabout 400 mg

Dosing can be once daily, or divided over multiple time periods duringthe day.

Effective dosing regimens can in part be established by measuring therate of excretion of the orally administered aminosterol and correlatingthis with clinical symptoms and signs. Exemplary dosing regimensinclude, but are not limited to: (1) Initiating with a “low” initialdaily dose, and gradually increasing the daily dose until a dose isreached that elicits evidence of the activation of the enteric nervoussystem, where the “low” dose is from about 10-about 100 mg per person,and the final effective daily dose is between about 25-about 1000mg/person; (2) Initiating with a “high” initial dose, which necessarilystimulates the enteric nervous system, and reducing the subsequent dailydosing to that required to elicit a clinically acceptable change inbowel behavior, with the “high” daily dose being between about 50-about1000 mg/person, and the subsequent lower daily oral dose being betweenabout 25-about 500 mg/person; (3) Periodic dosing, where an effectivedose can be delivered once every about 2, about 3, about 4, about 5,about 6 days, or once weekly, with the initial dose determined tocapable of eliciting an Aminosterol Induced Response.

Oral dosing should continue at least until the clinical condition hasresolved. To establish the need for continued dosing, treatment can bediscontinued and the condition reevaluated. If necessary, oraladministration should be resumed. The period of oral dosing can be forabout 1, about 2, about 3, or about 4 weeks; about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, or about 12 months, or about 1, about 2, about 3, about 4, or 5years, or longer.

In other embodiments of the invention, the first or initial “large” doseof squalamine (per person) can be selected from the group consisting ofabout 50, about 75, about 100, about 125, about 150, about 175, about200, about 225, about 250, about 275, about 300, about 325, about 350,about 375, about 400, about 425, about 450, about 475, about 500, about525, about 550, about 575, about 600, about 625, about 650, about 675,about 700, about 725, about 750, about 775, about 800, about 825, about850, about 875, about 900, about 925, about 950, about 975, about 1000,about 1025, about 1050, about 1075, about 1100, about 1125, about 1150,about 1175, about 1200, about 1225, about 1250, about 1275, about 1300,about 1325, about 1350, about 1375, about 1400, about 1425, about 1450,about 1475, about 1500, about 1525, about 1550, about 1575, about 1600,about 1625, about 1650, about 1675, about 1700, about 1725, about 1750,about 1775, about 1800, about 1825, about 1850, about 1875, about 1900,about 1925, about 1950, about 1975, and about 2000 mg. In otherembodiments of the invention, the second smaller dose of squalamine (perperson) is less than the first or initial dose and can be selected fromthe group consisting of about, 10, about 25, about 50, about 75, about100, about 125, about 150, about 175, about 200, about 225, about 250,about 275, about 300, about 325, about 350, about 375, about 400, about425, about 450, about 475, about 500, about 525, about 550, about 575,about 600, about 625, about 650, about 675, about 700, about 725, about750, about 775, about 800, about 825, about 850, about 875, about 900,about 925, about 950, about 975, and about 1000 mg. Finally, in otherembodiments of the invention, the periodic squalamine dosage (perperson) can be selected from the group consisting of about 10, about 25,about 50, about 75, about 100, about 125, about 150, about 175, about200, about 225, about 250, about 275, about 300, about 325, about 350,about 375, about 400, about 425, about 450, about 475, about 500, about525, about 550, about 575, about 600, about 625, about 650, about 675,about 700, about 725, about 750, about 775, about 800, about 825, about850, about 875, about 900, about 925, about 950, about 975, and about1000 mg.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes).

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of theaminosterol composition useful in the methods of the invention,including containers filled with an appropriate amount of a phosphate,either as a powder, to be dissolved, or as a sterile solution.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the aminosterol may be employed in conjunction with othertherapeutic compounds.

Pharmaceutical compositions according to the invention may also compriseone or more binding agents, filling agents, lubricating agents,suspending agents, sweeteners, flavoring agents, preservatives, buffers,wetting agents, disintegrants, effervescent agents, and otherexcipients. Such excipients are known in the art. Examples of fillingagents include lactose monohydrate, lactose anhydrous, and variousstarches; examples of binding agents include various celluloses andcross-linked polyvinylpyrrolidone, microcrystalline cellulose, such asAvicel® PH101 and Avicel® PH102, microcrystalline cellulose, andsilicified microcrystalline cellulose (ProSolv SMCC™). Suitablelubricants, including agents that act on the flowability of the powderto be compressed, may include colloidal silicon dioxide, such asAerosil® 200, talc, stearic acid, magnesium stearate, calcium stearate,and silica gel. Examples of sweeteners may include any natural orartificial sweetener, such as sucrose, xylitol, sodium saccharin,cyclamate, aspartame, and acesulfame. Examples of flavoring agents areMagnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors,and the like. Examples of preservatives include potassium sorbate,methylparaben, propylparaben, benzoic acid and its salts, other estersof parahydroxybenzoic acid such as butylparaben, alcohols such as ethylor benzyl alcohol, phenolic compounds such as phenol, or quaternarycompounds such as benzalkonium chloride.

Suitable diluents include pharmaceutically acceptable inert fillers,such as microcrystalline cellulose, lactose, dibasic calcium phosphate,saccharides, and/or mixtures of any of the foregoing. Examples ofdiluents include microcrystalline cellulose, such as Avicel® PH101 andAvicel® PH102; lactose such as lactose monohydrate, lactose anhydrous,and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®;mannitol; starch; sorbitol; sucrose; and glucose.

Suitable disintegrants include lightly crosslinked polyvinylpyrrolidone, corn starch, potato starch, maize starch, and modifiedstarches, croscarmellose sodium, cross-povidone, sodium starchglycolate, and mixtures thereof. Examples of effervescent agents includeeffervescent couples such as an organic acid and a carbonate orbicarbonate. Suitable organic acids include, for example, citric,tartaric, malic, fumaric, adipic, succinic, and alginic acids andanhydrides and acid salts. Suitable carbonates and bicarbonates include,for example, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium carbonate, sodium glycine carbonate,L-lysine carbonate, and arginine carbonate. Alternatively, only thesodium bicarbonate component of the effervescent couple may be present.

Combination Therapy. In the methods of the invention, the aminosterolcompositions may be administered alone or in combination with othertherapeutic agents. As noted above, the methods of the invention areuseful in treating and/or preventing the conditions described herein,including but not limited to chronic idiopathic constipation, Opioidinduced constipation, Irritable bowel syndrome, Inflammatory BowelDisease, Diabetes mellitus, Parkinson's disease, Alzheimer's disease,dementia of aging, Huntington's chorea, neuropathy of diabetes,peripheral sensory neuropathy, cerebral palsy, epilepsy, diabeticneuropathy, traumatic head and/or spine injury, stroke, Amyotrophiclateral sclerosis, multiple sclerosis, and certain malignancies. Thus,any active agent known to be useful in treating these conditions can beused in the methods of the invention, and either combined with theaminosterol compositions used in the methods of the invention, oradministered separately or sequentially.

For example, in methods of treating Irritable bowel syndrome, theaminosterol composition can be co-administered or combined with drugscommonly prescribed to treat IBS or related symptoms, such as alosetronhydrochloride (Lotronex®), fiber supplements or laxatives forconstipation or medicines to decrease diarrhea, such as diphenoxylateand atropine (Lomotil®) or loperamide (Imodium®). An antispasmodic iscommonly prescribed for treating IBS, which helps control colon musclespasms and reduce abdominal pain. Antidepressants may relieve somesymptoms of IBS. However, both antispasmodics and antidepressants canworsen constipation, so some doctors will also prescribe medicationsthat relax muscles in the bladder and intestines, such as belladonnaalkaloid combinations and phenobarbital (Donnatal®) and chlordiazepoxideand clidinium bromide (Librax®).

In methods of treating Inflammatory Bowel Disease, the aminosterolcomposition can be co-administered or combined with drugs commonlyprescribed to treat Inflammatory Bowel Disease or related symptoms, suchas aminosalicylates, corticosteroids, immune modifiers, anti-tumornecrosis factor (TNF) agents, and antibiotics. Exemplaryaminosalicylates include but are not limited to sulfasalazine(Azulfidine®), mesalamine (Asacol®, Pentasa®), olsalazine (Dipentum®),and balsalazide (Colazal®). Exemplary corticosteroids include but arenot limited to methylprednisolone, prednisone, prednisolone, budesonide,dexamethasone, hydrocortisone, betamethasone, cortisone, prednisolone,and triamcinolone. Exemplary immune modifiers include but are notlimited to 6-mercaptopurine (6-MP, Purinethol®) and azathioprine(Imuran®). An exemplary anti-TNF agent includes but is not limited toinfliximab (Remicade®). Exemplary antibiotics include but are notlimited to metronidazole and ciprofloxacin. Additional examples ofantibiotic agents include, but are not limited to, aminoglycosides,Ansamycins, Carbacephems, Carbapenems, Cephalosporins, Glycopeptides,Macrolides, Monobactams, Penicillins, Polypeptides, Polymyxin,Quinolones, Sulfonamides, Tetracyclines, and others (e.g., Arsphenamine,Chloramphenicol, Clindamycin, Lincomycin, Ethambutol, Fosfomycin,Fusidic acid, Furazolidone, Isoniazid, Linezolid, Metronidazole,Mupirocin, Nitrofurantoin, Platensimycin, Pyrazinamide,Quinupristin/Dalfopristin, Rifampicin (Rifampin in US), Thiamphenicol,Tinidazole, Dapsone, and lofazimine). Examples of these classes ofantibiotics include, but are not limited to, Amikacin®, Gentamicin®,Kanamycin®, Neomycin®, Netilmicin®, Streptomycin®, Tobramycin®,Paromomycin®, Geldanamycin®, Herbimycin®, Loracarbef®, Ertapenem®,Doripenem®, Imipenem®/Cilastatin®, Meropenem®, Cefadroxil®, Cefazolin®,Cefalotin® or Cefalothin®, Cefalexin®, Cefaclor®, Cefamandole®,Cefoxitin®, Cefprozil®, Cefuroxime®, Cefixime®, Cefdinir®, Cefditoren®,Cefoperazone®, Cefotaxime®, Cefpodoxime®, Ceftazidime®, Ceftibuten®,Ceftizoxime®, Ceftriaxone®, Cefepime®, Ceftobiprole®, Teicoplanin®,Vancomycin®, Azithromycin®, Clarithromycin®, Dirithromycin®,Erythromycin®, Roxithromycin®, Troleandomycin®, Telithromycin®,Spectinomycin®, Aztreonam®, Amoxicillin®, Ampicillin®, Azlocillin®,Carbenicillin®, Cloxacillin®, Dicloxacillin®, Flucloxacillin®,Mezlocillin®, Meticillin®, Nafcillin®, Oxacillin®, Penicillin®,Piperacillin®, Ticarcillin®, Bacitracin®, Colistin®, Polymyxin® B,Ciprofloxacin®, Enoxacin®, Gatifloxacin®, Levofloxacin®, Lomefloxacin®,Moxifloxacin®, Norfloxacin®, Ofloxacin®, Trovafloxacin®, Grepafloxacin®,Sparfloxacin®, Temafloxacin®, Mafenide®, Sulfonamidochrysoidine®(archaic), Sulfacetamide®, Sulfadiazine®, Sulfamethizole®,Sulfanilimide® (archaic), Sulfasalazine®, Sulfisoxazole®, Trimethoprim®,rimethoprim-Sulfamethoxazole® (Co-trimoxazole) (TMP-SMX),Demeclocycline®, Doxycycline®, Minocycline®, Oxytetracycline®, andTetracycline.

In methods of treating Diabetes mellitus, including both Type 1 and Type2 diabetes, or neuropathy of diabetes, the aminosterol composition canbe co-administered or combined with drugs commonly prescribed to treatDiabetes mellitus or related symptoms, such as insulin (ular and NPHinsulin, or synthetic insulin analogs) (e.g., Humulin®, Novolin®) andoral antihyperglycemic drugs. Oral antihyperglycemic drugs include butare not limited to (1) biguanides such as metformin (Glucophage®), (2)Sulfonylureas such as acetohexamide, chlorpropamide (Diabinese®),glimepiride (Amaryl®), Glipizide (Glucotrol®), Tolazamide, Tolbutamide,and glyburide (Diabeta®, Micronase®), (3) Meglitinides such asrepaglinide (Prandin®) and nateglinide (Starlix®), (4)Thiazolidinediones such as rosiglitazone (Avandia®) and pioglitazone(Actos®), (5) Alpha-glucosidase inhibitors such as acarbose (Precose®)and miglitol (Glyset®), (6) Dipeptidyl peptidase-4 inhibitors such asSitagliptin (Januvia®), (7) Glucagon-like peptide agonists such asexenatide (Byetta®), and (8) Amylin analogs such as pramlintide(Symlin®).

In methods of treating Parkinson's disease, the aminosterol compositioncan be co-administered or combined with drugs commonly prescribed totreat Parkinson's disease or related symptoms, such as levodopa (usuallycombined with a dopa decarboxylase inhibitor or COMT inhibitor),dopamine agonists and MAO-B inhibitors. Exemplary dopa decarboxylaseinhibitors are carbidopa and benserazide. Exemplary COMT inhibitors aretolcapone and entacapone. Dopamine agonists include, for example,bromocriptine, pergolide, pramipexole, ropinirole, piribedil,cabergoline, apomorphine, lisuride, and rotigotine. MAO-B inhibitorsinclude, for example, selegiline and rasagiline. Other drugs commonlyused to treat Parkinson's disease include, for example, amantadine,anticholinergics, clozapine for psychosis, cholinesterase inhibitors fordementia, and modafinil for daytime sleepiness.

In methods of treating Alzheimer's disease, the aminosterol compositioncan be co-administered or combined with drugs commonly prescribed totreat Alzheimer's disease or related symptoms, such as Glutamate,Antipsychotic drugs, Huperzine A, acetylcholinesterase inhibitors andNMDA receptor antagonists such as memantine (Akatinol®, Axura®,Ebixa®/Abixa®, Memox® and Namenda®). Examples of acetylcholinesteraseinhibitors are donepezil (Aricept®), galantamine (Razadyne®), andrivastigmine (Exelon®).

In methods of treating Huntington's chorea, the aminosterol compositioncan be co-administered or combined with drugs commonly prescribed totreat Huntington's chorea or related symptoms, such as medicationsprescribed to help control emotional and movement problems associatedwith Huntington's chorea. Such medications include, but are not limitedto, (1) antipsychotic drugs, such as haloperidol and clonazepam, (2)drugs used to treat dystonia, such as acetylcholine-regulating drugs(trihexyphenidyl, benztropine (Cogentin®), and procyclidine HCl);GABA-regulating drugs (diazepam (Valium®), lorazepam (Ativan®),clonazepam (Klonopin®), and baclofen (Lioresal®)); dopamine-regulators(levodopa/carbidopa (Sinemet®), bromocriptine (parlodel)), reserpine,tetrabenazine; anticonvulsants (carbamazepine (Tegretol®); and Botulinumtoxin (Botox®); and (3) drugs used to treat depression (fluoxetine,sertraline, and nortriptyline). Other drugs commonly used to treat HDinclude amantadine, tetrabenazine, Dopamine blockers, and co-enzyme Q10.

In methods of treating peripheral sensory neuropathy, the aminosterolcomposition can be co-administered or combined with drugs commonlyprescribed to treat peripheral sensory neuropathy or related symptoms.Peripheral sensory neuropathy refers to damage to nerves of theperipheral nervous system, which may be caused either by diseases of ortrauma to the nerve or the side-effects of systemic illness. Drugscommonly used to treat this condition include, but are not limited to,neurotrophin-3, tricyclic antidepressants (e.g., amitriptyline),antiepileptic therapies (e.g., gabapentin or sodium valproate),synthetic cannabinoids (Nabilone) and inhaled cannabis, opiatederivatives, and pregabalin (Lyrica®).

In methods of treating traumatic head and/or spine injury, theaminosterol composition can be co-administered or combined with drugscommonly prescribed to treat traumatic head and/or spine injury orrelated symptoms, such as analgesics (acetaminophen, NSAIDs,salicylates, and opioid drugs such as morphine and opium) andparalytics.

In methods of treating stroke, the aminosterol composition can beco-administered or combined with drugs commonly prescribed to treatstroke or related symptoms, such as aspirin, clopidogrel, dipyridamole,tissue plasminogen activator (tPA), and anticoagulants (e.g., alteplase,Warfarin, dabigatran).

In methods of treating Amyotrophic lateral sclerosis, the aminosterolcomposition can be co-administered or combined with drugs commonlyprescribed to treat Amyotrophic lateral sclerosis or related symptoms,such as riluzole (Rilutek®), KNS-760704 (an enantiomer of pramipexole),olesoxime (TRO19622), talampanel, Arimoclomol, medications to helpreduce fatigue, ease muscle cramps, control spasticity, reduce excesssaliva and phlegm, control pain, depression, sleep disturbances,dysphagia, and constipation.

In methods of treating multiple sclerosis, the aminosterol compositioncan be co-administered or combined with drugs commonly prescribed totreat multiple sclerosis or related symptoms, such as corticosteroids(e.g., methylprednisolone), plasmapheresis, fingolimod (Gilenya®),interferon beta-1a (Avonex®, CinnoVex®, ReciGen® and Rebif®), interferonbeta-1b (Betaseron®, Betaferon®), glatiramer acetate (Copaxone®),mitoxantrone, natalizumab (Tysabri®), alemtuzumab (Campath®), daclizumab(Zenapax®), rituximab, dirucotide, BHT-3009, cladribine, dimethylfumarate, estriol, fingolimod, laquinimod, minocycline, statins,temsirolimus teriflunomide, naltrexone, and vitamin D analogs.

In methods of treating cerebral palsy, the aminosterol composition canbe co-administered or combined with drugs commonly prescribed to treatcerebral palsy or related symptoms, such as Botulinum toxin Ainjections.

In methods of treating epilepsy, the aminosterol composition can beco-administered or combined with drugs commonly prescribed to treatepilepsy or related symptoms, such as anticonvulsants (e.g.,carbamazepine (Tegretol®), clorazepate (Tranxene®), clonazepam(Klonopin®), ethosuximide (Zarontin®), felbamate (Felbatol®),fosphenytoin (Cerebyx®), gabapentin (Neurontin®), lacosamide (Vimpat®),lamotrigine (Lamictal®), levetiracetam (Keppra®), oxcarbazepine(Trileptal®), phenobarbital (Luminal®), phenytoin (Dilantin®),pregabalin (Lyrica®), primidone (Mysoline®), tiagabine (Gabitril®),topiramate (Topamax®), valproate semisodium (Depakote®), valproic acid(Depakene®), and zonisamide (Zonegran®), clobazam (Frisium®), vigabatrin(Sabril®), retigabine, brivaracetam, seletracetam, diazepam (Valium®,Diastat®), lorazepam (Ativan®), paraldehyde (Paral®), midazolam(Versed®), pentobarbital (Nembutal®), acetazolamide (Diamox®),progesterone, adrenocorticotropic hormone (ACTH, Acthar®), variouscorticotropic steroid hormones (prednisone), and bromide.

In methods of treating depression, the aminosterol composition can beco-administered or combined with drugs commonly prescribed to treatdepression, such as any of the class of Tricyclic antidepressants,Monoamine oxidase inhibitors, Selective serotonin reuptake inhibitors,and Serotonin and norepinephrine reuptake inhibitors.

In the methods of treating malignancies, the aminosterol composition canbe co-administered or combined with drugs commonly used to treatmalignancies. These include all known cancer drugs.

Combinations may be administered either concomitantly, e.g., as anadmixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second. The regimen selected can be administeredconcurrently since activation of the aminosterol induced response doesnot require the systemic absorption of the aminosterol into thebloodstream and thus eliminate concern over the likelihood systemic ofdrug-drug interactions between the aminosterol and the administereddrug.

The following examples are provided to illustrate the present invention.It should be understood, however, that the invention is not to belimited to the specific conditions or details described in theseexamples. Throughout the specification, any and all references to apublicly available document, including a U.S. patent, are specificallyincorporated by reference.

EXAMPLES Example 1

The purpose of this example was to evaluate the pharmacological effectof squalamine/Aminosterol 1436 administration on gastrointestinalbehavior, referred to as “the Aminosterol Induced GI Response.”

Gelatin capsules were prepared for oral administration. Capsules werecoated in shellac to prevent their release within the stomach.Furthermore, because squalamine and related aminosterols have antibioticactivity, enteric coating prevents the drug from altering the microbialpopulations of the stomach. The proximal small intestine, where thecapsules should dissolve, is normally nearly sterile and hence notsignificantly perturbed microbiologically by the presence of theaminosterols. Squalamine dilactate powder, 99% pure, or 1436hydrochloride, 99% pure, was added manually into either Size 0 (for the200 mg dose) or Size 1 (for the 100 mg dose) gelatin capsules. Noexcipient was added. The capsules were coated twice in 5% shellac (80%acetone/20% ethanol) and dried before use.

200 mg of squalamine (lactate salt, excipient free) in a coated gelatincapsule was administered orally to a human male in the morning on anempty stomach, with water. Squalamine was synthesized as described inZasloff et al 2011. Within 2 hours nausea was experienced, lasting about30 minutes. At 2.5 hours, increased peristalsis (“rumbling gut”) wasexperienced, lasting several minutes. At about 3 hours, a clear waterydischarge of about 200 ml was passed rectally. A second episode occurredat 3.2 hours. Following this episode, bowel sounds quieted. No nausea ordiscomfort was subsequently experienced. Appetite was near normal.Despite normal feeding, feces were not passed for 2.5 days over whichtime no discomfort was experienced. The first feces passed after thisquiescent period was soft and relatively small considering the intake offood and the time interval. Usual bowel functions resumed by about 4days after squalamine administration.

Results: Since the normal bowel behavior of this individual is about 1passage every 24 hours, it can be estimated that 200 mg of squalaminelactate reduced normal small intestinal motility by 2.5 fold. The normalconsistency of the stool, despite the delay in passage, reflects acompensatory physiological adjustment in intestinal fluid secretion andabsorption, and colonic motility, sufficient to maintain normal moisturecontent of the fecal material.

Example 2

The purpose of this example was to evaluate the pharmacological effectof squalamine/Aminosterol 1436 administration on gastrointestinalbehavior.

200 mg of 1436 (HCl salt, excipients free) in a gelatin capsule wasadministered orally to a human male in the morning on an empty stomach,with water. Within 1 hour increased peristalsis was experienced, withoutnausea. At 2 hours, about 100 ml of clear watery diarrhea, recurring at2.75 hours, 3 hours, 3.25 hours, and 4 hours, with progressively lesservolumes of liquid discharged. First bowel movement was passed at 3.5days, of normal consistency. The subject experienced no abdominaldiscomfort, bloating, etc over this period. Usual bowel functionsresumed by about 4.5 days after administration of Aminosterol 1436.

Based on this study, it can be estimated that 200 mg of Aminosterol 1436(HCl salt) has reduced small intestinal motility by 3.5 fold. As notedfollowing the administration of squalamine, the first passed stool wasof normal consistency despite the delay in passage.

Example 3

The purpose of this example was to evaluate the pharmacological effectof squalamine/Aminosterol 1436 administration on gastrointestinalbehavior.

200 mg of squalamine (lactate salt, excipient free) in a gelatin capsulewas administered orally to a human male in the evening following a largemeal. No gastrointestinal response related to the administration ofsqualamine could be perceived either overnight or by the followingmorning. A normal bowel movement was passed in the morning.

This response suggests that optimal oral dosing should be on an emptystomach. In addition, this observation suggests that squalamineinitiates the Aminosterol-Induced GI Response in the proximal smallintestine rather than in the distal. Squalamine, because of its physicalproperties, would be expected to bind tightly to foodstuff, and beunavailable to interact with the intestinal epithelium.

Example 4

The purpose of this example was to evaluate the pharmacological effectof squalamine/Aminosterol 1436 administration on gastrointestinalbehavior.

200 mg of squalamine was administered to a human male as in the firstexample. All stool was collected for 6 days following administration.Stool was then extracted with 60% acetonitrile and 1% HCl overnight. Thesupernatant was collected and the presence of squalamine assessed bythin layer chromatography following published procedures. Approximately80% of the dose could be recovered from the stool.

Considering losses and efficiencies of extraction, and known lowbioavailability of squalamine in mice and dogs, it can be concluded thatthe effects observed on the GI tract ultimately resulted from directinteractions between squalamine and gut epithelial and neuronal cells.

Example 5

The purpose of this example was to evaluate the pharmacological effectof squalamine/Aminosterol 1436 administration on gastrointestinalbehavior.

100 mg of squalamine was orally administered in a capsule as in thefirst example. Neither nausea, nor watery diarrhea was experienced. Aperiod of “bowel quiet” was induced, however, that lasted 2 days, afterwhich normally formed feces were passed.

These results teach that the reduction in bowel motility can occur atlower doses of squalamine without the conscious sensation of nausea andthe appearance of diarrhea. The soft consistency of the stool, despiteits delayed transit time, suggests that luminal fluid exchange in thesetting of the Aminosterol-Induced GI Response is physiologicallymatched to accommodate the reduction in small intestinal motility.

Example 6

The purpose of this Example was to determine whether orally administeredsqualamine, at a dose sufficient to provoke the completeAminosterol-Induced GI Response, would stimulate release of GLP-1 intothe blood stream.

Enteroendocrine cells, which contain entreric hormones, are known torespond to a variety of stimuli they contact in the intestinal lumen,and release hormones into the submucosa and subsequently into thebloodstream. In particular, GLP-1, the product of the L-cell, is knownto have motility effects on the small intestine that resemble thoseobserved after oral administration of squalamine. In this example, alipid/carbohydrate meal was administered which is known to provoke therelease of GLP-1 into the bloodstream. The GLP-1 response of the GItract to the meal was compared in the presence or absence of squalamine,with blood levels of GLP-1 measured using an ELISA based immunoassay.

The basic protocol was as follows:

Control Response

-   1. Subject (Caucasian male, 66 yrs, BMI: 2.2, no medical illness)    was fasted overnight-   2. IV catheter 18 gauge implanted in R decubitus fossa-   3. Blood samples drawn at 9:30 A, 11 A, 11:30 A, 12:30 P, 1:30 P, 2    P, 2:30, 3, 3:30, 4:30, 5:30-   4. Empty capsule and water at 10:30 A-   5. Lipid/carbohydrate meal (2 slices of white bread, each with 5.5    gms butter) at 2 P-   6. Blood samples: 2 ml into Becton-Dickenson Protease-inhibited    Biomarker tubes; 3 ml into standard red top blood collection tubes.-   7. All samples were immediately spun at 3000 rpm×15 and serum or    plasma aliquotted into 1.5 ml eppendorfs and then stored at −80° C.    Squalamine Response-   1. Subject rested 3 days and re-evaluated 4 days after the Control    response-   2. Fasted overnight-   3. IV catheter implanted-   4. Samples drawn at 10, 11, 11:30, 12:30, 1:30, 2:30, 3, 3:30, 4:30,    5:30-   5. Squalamine capsule, 200 mg at 10:30 A-   6. Bread and butter, 2 P-   7. Blood samples collected and processed as in the Control study

Outcomes:

Squalamine Response: Clinical

Episodes of light headedness 11 A-12:15

Episode of mild nausea 1:45

Passage of watery diarrhea: 2 P, 2:20, 2:36, 3:15,

GLP-1 analyses (Total immunoreactive GLP-1): A Millipore ELISA kit wasused (Cat #EZGLP1T-36K). A standard curve, provided in the kit,demonstrated linear response between 0→20 pM. The results are shown inTable 1. No significant effect of squalamine on the release of GLP-1from the small intestine was observed.

TABLE 1 Control GLP-1(pM) Squalamine GLP-1(pM0  9:30 6 10 6 11 11 11 511:30 12 11:30 4 12:30 17 12:30 10  1:30 7  1:30 9  2:30 11  2:30 8 3:00 12  3:00 8  3:30 1  3:30 6  4:30 5  4:30 5  5:30 10  5:30 3

This Example demonstrates that orally administered squalamine does notpharmacologically stimulate secretion of Enteroendocrine cellsexpressing GLP-1, and that the effects on bowel motility cannot be aconsequence secondary to the presence of elevated levels of GLP-1.

Example 7

The purpose of this example was to evaluate the effects of squalamine,orally dosed, on Irritable Bowel Syndrome (IBS).

A 66 year old female, in otherwise good health, was suffering from the“mixed” form of IBS since early childhood. The condition wascharacterized by a failure to feel “cleared” after the passage of astool; occasional crampiness; abdominal bloating and a sense of“fullness.” The condition caused her to be “conscious of her bowels”throughout the day, and frequently interrupted her sleep. Attempts torelieve herself on the toilet would generally be ineffective, leavingher with the feeling of “still having to go.”

On day 1 the individual was administered a single 200 mg capsule ofsqualamine, orally, on an empty stomach with water. Within 2 hrs sheexperienced nausea, at 3 hrs she vomited, followed soon after by thepassage of about 200 ml of clear discharge rectally. By 3.5 hrs thenausea had passed. She ate normally at lunch and dinner. She experienced“bowel quiet” from Day 1 through Day 5 on a single dosing, withoutcramping or urgency. Slept better than she had in many years. The firststool was passed on Day 4, was soft and passed easily, and thensuccessively on Days 5, 6, 7, and 8. Beginning at Day 6, she began toexperience the prior GI symptoms. By Day 8, her usual GI symptoms hadreturned.

A second dosing trial was begun on Day 15, with a 50 mg capsuleadministered. Neither nausea nor a discharge was experienced. Sense ofbowel quiet was experienced. Bowel movement was passed on Day 17. A 25mg capsule was administered on Day 19. By Day 21 the previous symptomsof IBS were returning. It was assumed that 50 mg dosed every other daywas an optimum regimen. The individual remained on a dosing schedule of50 mg orally every other day.

At 10 months of dosing the individual no longer experiences symptoms ofIBS. Bowel movements are of normal consistency. Sleep is no longerinterrupted by urgency to defecate. Individual believes that thetreatment has resulted in improvement of mood and responsible for a“sense of well-being.” Stools are somewhat smaller than previouslynoted, and passed generally once daily.

This Example demonstrates the utility of the invention for the treatmentof Irritable Bowel Syndrome and establishes an effective dosing regimen.

Example 8

The purpose of this example was to evaluate the effects of squalamine,orally dosed, on the constipation associated with Parkinson's disease.

A 70 yr old male, in otherwise good health, presented with Parkinson'sdisease of about 5 yrs duration. He was severely constipated andsuffered from frequent episodes of cramping. These GI symptoms weresimilar to that described in Parkinson's and could not be alleviatedmedically or through dietary adjustment. His medications included 5L-DOPA/DDC caps/day and 1-2 Requip before bedtime. His neurologicalstatus at this time was as follows, as determined about 1 hour after anL-DOPA dosing, a time point when he appears to be repleted withdopamine:

-   -   Slow walking with shuffling . . . need to squat intermittently    -   Articulation poor . . . difficult to understand his speech . . .        halting    -   Use of utensils while eating involved slow, awkward movements    -   Face seemed a bit mask like; unconsciously made chewing        movements    -   Cognitive function (memory, wit) was less than observed 3 months        prior    -   Balance was unsteady    -   Mood was depressed    -   Swallowing noted to be difficult at times    -   Chewing food was slow    -   Handwriting was small

Began oral dosing with 200 mg capsule of squalamine on Day 1. Capsulewas taken in the AM before breakfast, along with L-DOPA. No nauseaexperienced nor watery discharge. Passed several moist stools on Day 1.200 mg cap on day 2 provoked a watery diarrhea at 2 hrs after dosing,but discharge subsided by day 3 dosing. On day 13, subsequent dosing wasmaintained at 200 mg every other day, with some minor degree of nausea.No watery discharge. At about Day 30 the subject was switched to 100 mgevery other day, and remained on this regimen for about 9.5 months.

The therapeutic response to administration of squalamine was progressiveand objectively apparent, as detailed in Table 2

TABLE 2 Time Point Observations 3 Weeks Bowels improved considerably(Observations Walking more comfortably, but still uneven were collectedSubject feels breathing is easier over 3 Articulation much improveddays) Able to touch the back of front upper teeth with his tongue Moodseems to have improved Some shaking even after L-DOPA Balance still poorin the AM, but some improvement observed 2 Months Normal bowelfunctions; no (Observations constipation; no GI issues; no werecollected cramping over 5 No shaking of arms and legs days) Walking moresmoothly, arms swinging more naturally Increased stamina Subject couldhold various objects in his hand while walking a block or two or walkingup stairs; unlike previously, now appeared confident in being able tohold objects while he walked Could sustain concentrated work whichinvolved sorting through papers, bending over to pick up and movepapers; he did this for 2.5 hours without stopping. Until now hisability to sustain this moving and bending was very limited such that hereported pain in his back and general weakness that shortened the worksessions to less than one hour. Subject's ability to handle utensilswhen he eats was much improved; he could control the fork and spoon in anatural way which was not the case a few months ago. The speed of hisability to move the utensils was very normal looking now as opposed tothe very slow pace at which he moved utensils over the past months.Subject still reports periods when he feels very tired. However over the5 days of observation as opposed to the previous visit, he did not needto squat down to relieve his legs from feeling exhausted. He walkedwithout shuffling His speech is now smooth and normal in volume andarticulation. This was not the case months ago. He did not report havingdifficulty swallowing now, as opposed to before. He was able to talk andeat at the same time. He has retained an oral mannerism in which hechews his tongue/cheek. This has developed over the past few months. Heis aware that he is doing it and reports it has become a habit. Subjectdid not make any extreme mannerisms of the face that had begun to beseen over the past few months Subject has expressed hopefulnessregarding the regaining of strength and a general feeling of betterhealth. This hopefulness is new. Though historically an anxiouspersonality, Subject seems less so now. Seems more capable ofcontrolling anxiety and making positive decisions: moving out of hisoffice, packing, disposing of unnecessary property, papers, etc. Themost evident Parkinson's disease sign that remained was instability whenbeing pushed or pulled. He would lose balance and compensate by movingin the direction of the force with faltering choppy steps, along withvocalizing “Oh, Oh, Oh . . . ” Medication needs remained stable. 9months Bowel functions normal (Observations Generally, within about anhour were collected after taking an L-DOPA dose, over 5 Subject does nothave the appearance days) or behavior, or signs of a person withParkinson's disease. Balance, as tested in the previous visit, by pushor pull was met with resistance on his part, opposing applied force.Subject remained upright and stable, not moving from his standingposition. All movements (hands, walking,) are all smooth and normal. Noshaking noted Walking is strong without any evidence of shuffling. Wasable to walk on even terrain in the course of a hike through Will RogersPark, lasting about 2 hours. Was then eager to continue on through avisit to the Getty Museum. No fatigue. Excellent stamina. Mood has beenupbeat Articulation normal Handwriting seems normal Cognitive functionsnormal. Memory normal. Medication needs have remained stable: 4L-DOPA, + 1-2 Requip/day.

This Example suggests that orally administered squalamine hastherapeutic benefit in Parkinson's disease and establishes an effectivedosing regimen. The assessment is that there appears to be 2 potentialtargets. The first is the dopamine producing cells within the substantianigra. The second is the functionality of the nervous system circuitry.Squalamine appears to have restored function to the nervous system,permitting near normal functioning when the subject is replete withdopamine (administered exogeneously). The aminosterol dosing regimen maypossibly have stopped further deterioration of the dopamine producingtissues, or perhaps even restored some addition production capacity, butif so this effect is much less apparent than the recovery of overallneurological behavior.

The progression of nervous system recovery appears to begin with the GItract, the target being affected most likely the enteric nervous system.Constipation resolved within several weeks. A bit later, improvement inarticulation was noticed. Arm and leg movement follow. Last to improveis balance.

Example 9

The purpose of this example was to evaluate the effects of squalamine,orally dosed, on the neurological signs associated with Parkinson'sdisease in an individual without the usual Parkinson's associatedconstipation and GI disorders and likely a “Parkinson's-like” syndrome.

The individual was a male, 59 years old. He was a psychiatristspecializing in addiction. Movement disorder began at age 53. Originaldiagnosis was multiple sclerosis. Presenting symptoms were depressionand difficulty in controlling the action of sitting in chair. He would“plop” onto a sit without braking the movement. An MRI showed manydiffuse lesions scattered throughout the cortex, which appear to haveremained stable. Treatment with L-DOPA caused some correction of thesitting difficulty, leading to the presumptive diagnosis of Parkinson'sdisease. Therapy continued with increasing doses of Sinemet, followed byaddition of ropinirole. Deterioration continued (despite an L-DOPA doseexceeding 10-11 pills/day), ropinirole (5×2 mg and 4×1 mg, daily) andamantadine was added. He was on Wellbutryn for depression. There hadbeen no history of GI disorders, and bowel function was said to benormal.

The individual lived at home with a wife and 2 children. He had to bedriven to work. Walking was unsteady. His speech was nearlyincomprehensible. Face was mask-like. No obvious tremors. Walking washesitant. Subject had fallen (or tripped) several times previously andused a cane. Table 3 summarizes the dosing protocol and observations.

TABLE 3 Timing Observations Day 1 Subject took a 100 mg cap ofsqualamine. No obvious GI response. Continued on 100 mg every other day.Day 9 Observed Subject walking smoothly and briskly from a distancewithout walker or cane. Day 14 Subject sounded more coherent thanpreviously. This was noted, as well by his patients. Day 21 Individualindependently began 200 mg every other day. Others noted that hisarticulation had become noticeably better. His self-confidence was notedto be improving and his energy level was increasing. Month 3 Subject ranout of squalamine 2 weeks prior. Cessation was followed by “the worstconstipation he had ever remembered.” This resolved spontaneously overthe following few days. He also noted that he “was not feeling as good”as when on it . . .” not exactly depression. hard to explain . . .” Hisarticulation was clear. Month 4 Subject began 100 mg every other day.Discovered that his family situation was extremely unstable and he wasanticipating a divorce or separation. He stopped taking L-DOPA on aregular basis but continued on squalamine. Month 5 Subject independentlyraised dose to 200 mg every day at month 5.5. No GI issues (i.e.,diarrhea). Commented that colleagues noted improvement. Took severalfalls which he attributed to “risky behavior” on his part. Month 6Individual could compensate being pulled and pushed without faltering.He told me that he felt his balance had improved. Still had difficultyinitiating walking. No tremor. Voice was of low volume. Face was stillmask-like. Once moving subject walked without shuffling. Subject wasstill having very complex family issues. Steadfastly insisted oncontinuing squalamine. Medication dosing had not changed.

This Example suggests that squalamine can provide therapeutic benefit inindividuals with a Parkinson's like syndrome in whom no initialgastrointestinal symptoms are noted.

Example 10

An experiment was conducted in the mouse to test the hypothesis thatsqualamine was acting on the enteric nervous system, as predicted by theclinical responses observed in human subjects.

Under appropriate and approved anesthesia, the distal colon of a mousewas externalized from the abdomen, along with the mesentary. Themesenteric blood vessels and lymphatics were carefully dissected fromthe mesenteric nerve, a structure that contains both the vagus (efferentand afferent) and spinal afferents. The nerve was cut, and the free end(which receives signals from the gut) was introduced into a capillaryhousing a silver electrode, along with the appropriate electronicsrequired to detect and amplify weak electrical signals pulsing from thegut through the mesenteric nerve. As seen in FIG. 1, introduction ofsqualamine into the lumen of the colon results in an increase inelectrical activity directed toward the brain from the gut nervoussystem. Concentrations of 3, 10, 30 and 100 μM squalamine wereevaluated. Responses to squalamine application were discernible at 30 μMbut robust and reproducible responses were first evident at 100 μM. Atthe latter concentration, 5 experiments were successfully performed andeach revealed that squalamine evoked an increase in the mesenteric nervemultiunit firing frequency. Paired experiments were comparisons ofaverage discharge rates of a control recording period with athree-minute period during the peak response to squalamine. Statisticalcomparisons were made using paired t-tests. Squalamine increased thebackground firing rate from (mean±SD) 2.0±1.2 to 5.2±3.1 Hz (P=0.03).The latency to the onset of the firing increase was 4.4±4.1 min, whilethe latency to peak of the firing rate increase 12±7.5 min (FIG. 1).

This Example teaches that squalamine, administered to the gut lumen,stimulates enteric neurons, and that some of these neurons can sendcurrents to centers in the CNS. This supports the utility of orallyadministered squalamine in those conditions in which stimulation ofafferent currents within the vagus could provide therapeutic benefit.

Example 11

An experiment was performed to determine the effect of squalamine oncolonic motility and intraluminal pressure.

A segment of colon was excised from a mouse, and one end was attached toa glass tube housing a pressure sensor. A video camera recorded thevisible contractions of the segment. When squalamine was added to thecolonic lumen, the frequency of contractions increased about 2 fold,representing coordinated peristaltic waves that move toward the rectum.The peak pressure of these waves did not differ from those observedprior to squalamine application. The data are shown in FIG. 2.

This Example demonstrates that squalamine has “anti-constipation”properties. This animal model is used to demonstrate, for example, theconstipation inducing properties of opioid, such as loperamide. ThisExample supports the utility of squalamine in human disorderscharacterized by delayed colonic motility.

Example 12

An experiment was conducted in the mouse to determine whether squalaminecan stimulate the instrinsic primary afferent neurons (IPANs) of thecolon, the neurons that act within the colon to influence motility andsecretion.

In this experiment the outer adventitial layer of the mouse colon ispeeled away exposing the IPANs. Individual IPANs within the myentericplexus of the wall of the colon are patched using a patch-clampapparatus. These cells represent the major neuronal population thatcommunicate sensory data from the gut. They synapse with the variousnerve networks within the gut that influence muscle and secretoryactivity, as well as connecting with vagal nerve endings. Administrationof squalamine at 30 μM to the lumen (applied via a 50 ms “puff” ofsolution) leads to a very sustained (10 min) burst of electricalactivity (FIG. 3).

This Example shows that the nerve ending of these key sensory cells,which lie near the luminal surface of the colon, can detect squalamineand respond electrically with robust, long lived signals. Moreover, thisExample shows that squalamine's effect on intestinal motility and musclecontraction (and possibly secretion) are likely a consequence secondaryto stimulation of the IPANs, which in turn communicate to the myentericand submucosal plexus.

Example 13

An experiment was conducted to determine whether squalamine stimulatedthe IPANs directly or indirectly.

In this Example the same procedure as that described in Example 12 wasfollowed except that the epithelial lining of the colon was peeled awayprior to application of squalamine to the lumen. Signals from individualIPANs were recorded. Concentrations of squalamine as low as 1 uM couldnow stimulate a robust response, similar quantitatively (andqualitatively) to concentrations 30-100 fold higher, when an intactepithelium was present.

This Example clearly shows that squalamine interacts directly with theneurons of the intestine to stimulate activity. If the compound actedindirectly on the nervous system, i.e., by stimulating enterochromaffincells to release neutrotransmitters such as serotonin or histamine,which, in turn, stimulated their receptors on the nerve cells, strippingof the epithelium would have caused the opposite result, namely the lossof squalamine responsiveness. This Example supports the utility ofsqualamine in the treatment of diseases of the nervous system wherereduction in the net surface potential of the intracellular membranecould elicit benefit, such as in the displacement of alpha-synucleinfrom the plasma membrane of neurons in Parkinson's disease.

Example 14

The purpose of this Example was to demonstrate the capacity ofsqualamine to displace alpha-synuclein from membranes that exhibit anegative surface potential.

Alpha-synuclein was mixed with a preparation of vesicles consisting ofDOPE (50%), DOPS (30%), DOPC (20%). The concentration of lipid was 2.4mM; the concentration of N-terminally acetylated alpha-synuclein was 10μM. The interaction was followed by circular dichoroism. Theconcentration of squalamine was increased in 40 μM increments. As theconcentration of squalamine increased, a linear decrease in the amountof alpha-synuclein membrane bound was observed, reflecting the higheraffinity of squalamine than alpha synuclein for the negatively chargedphospholipids. At a ratio of lipid to squalamine of 15:1, fulldisplacement of alpha-synuclein was observed. Addition of squalamine toa solution of alpha-synuclein alone had no effect on the circulardichroism spectrum.

This Example demonstrates that squalamine has the capacity to physicallyreduce the concentration of membrane bound alpha-synuclein. Hence, thisExample supports the utility of administering squalamine and relatedaminosterols in conditions where alpha-synuclein/membrane interactionsare believed to cause pathology, such as in Parkinson's disease.Beta-Amyloid, the protein associated with Alzheimer's, like alphasynuclein, is known to bind to anionic phospholipids; from the resultsof this Example, it would be logical to assume that squalamine and otherrelated aminosterols should displace Beta-amyloid from its targetmembranes, supporting its utility in the treatment of Alzheimer'sdisease.

Example 15

An experiment was conducted to determine whether squalamine exhibitedefficacy in the treatment of Opioid induced constipation.

A normal subject began dosing with 1 tab codeine 30 mg/acetaminophen 300mg at 8 A on Day 1, and repeated on Day 2 and Day 3. Normal bowelmovement frequency was 1 or more stools/day, normally formed. Prior tocodeine on Day 1, subject passed a normal stool in the AM. No bowelmovements from the start of dosing through the 8 A Day 3 dose. Nocramping; some gas. Some sense of fullness on the evening of Day 2.

At 9:15 A subject was administered 200 mg cap of squalamine. Withinabout 10 min the subject had a cup of coffee and some cookies. Nonausea. No clear rectal fluid. At 11:15 A, the subject passed a largesoft, well formed stool with normal urgency. A second smaller stool waspassed at 12:30. A third, large watery stool was passed at 7 P. A senseof bowel relief was appreciated by the subject. The rapid effect oncolonic motility seems unlikely to have been a direct effect ofsqualamine on the colon, but perhaps a downstream consequence of entericnervous system stimulation. On Day 4, 30 mg codeine. No squalaminedosing. 7 P passed a loose stool of normal volume.

This Example suggests that the Aminosterol-Induced GI Response canover-ride the inhibitory effects of opioids on GI function. Moreover,this Example provides evidence that squalamine and related aminosterolscan exert therapeutic benefit in the setting of opioid-inducedconstipation.

Example 14

The purpose of this example was to demonstrate the utility ofstimulating the Aminosterol-Induced GI Response to induce competeregression in untreatable, unresectable Stage 4b colon cancer.

A 65 year old white male presented with abdominal distention, pain, andprogressive weight loss of several months duration. Radiographic studiesrevealed widely disseminated masses throughout the abdomen. Anexploratory surgical procedure revealed widespread implants of amucinous adenocarcinoma, throughout the peritoneum, on and within theliver, and covering the other major organs without a clear primarytumor. Because of the extent of the cancer, the decision to close wasmade and no further treatment other than symptomatic management and endof life care was suggested.

The individual was begun on a total daily dose of 50 mg of squalamine,administered as 25 mg twice daily, as a powder dispersed in apple sauce.Approximately 6 months after initiation of squalamine treatment theindividual was seen again at the hospital due to the chance recognitionof a mass on his right kidney. The kidney was surgically removed, andduring the procedure the peritoneum explored. No tumor was visible. Themass on the kidney was a benign cyst. Treatment with squalaminecontinued daily for 5 years. The individual had no recurrence of cancer.The subject died at age 83 of a myocardial infarction.

Example 15

The purpose of this example was to demonstrate the utility ofstimulating the Aminosterol-Induced GI Response to induce regression ofendometrial cancer.

A 66 year old white female presented with abdominal discomfort and thepresence of a palpable abdominal mass. Radiographic studies revealed thepresence of a uterine mass and numerous affected nodes extending fromsupraclavicular to peritoneal. Surgical removal of the uterus andovaries was followed by chemotherapy. Subsequently, a large tumoradherent to the distal colon was discovered, along with additionalnodes. A colonic resection was performed along with local radiation tothe peritoneum.

The individual decided to stop all chemotherapy due to progression ofthe cancer and the adverse reactions previously experienced. The subjectbegan oral squalamine in capsule form in August 2012. By March 2013, noevidence of malignancy was visible by PET/CT imaging. In July 2013 asmall tumor appeared adjacent to the right ureter, and a second attachedto the left descending colon. The dose of squalamine was raised to 100mg/day in two divided doses. In October 2013, the tumor adjacent to theureter was irradiated. In February 2013, the small mass adherent to thecolon remained active by PET/CT, slightly larger than when first notedin July 2013, and no additional metasteses were observed. The individualremains on squalamine 100 mg/day. She is in otherwise excellent health.

Example 16

The purpose of this example was to demonstrate the utility ofstimulating the Aminosterol-Induced GI Response to induce regression ofStage 4b pancreatic cancer

A 67 year old white female presented with abdominal discomfort, weightloss, and malaise. After considerable medical consultations a diagnosisof pancreatic adenocarcinoma was made. The subject was treated withchemotherapy and experienced a severe adverse reaction, which includedascites and pleural effusion. In July 2013, a PET/CT revealed thepresence of a mass in the head and tail of the pancreas along withseveral tumors in the liver and numerous active peripheral lymph nodes.In August 2013, the subject was evaluated for a clinical trial at TGENin Arizona, but not admitted due to the extensive cancer evident in thepancreas, liver, and nodes.

In late August 2013, the subject was begun on 50 mg/day, then increasedto 100 mg/day after a week. The subject began once monthly cycles ofcisplatin and 5-FU. A PET/CT scan in early December 2013 revealed thedramatic reduction in PET intensity of all previously active cancer. APET/CT scan of February 2014 revealed the complete loss of all PETactive masses, including the lymph nodes. The overall level ofperformance of the individual continues to improve, but the ascites andpleural effusions persist. Cytology of the peritoneal fluid remainsnegative for abnormal cells. All concurrent chemotherapy has stopped.Squalamine treatment continues.

Example 17

The purpose of this example was to demonstrate the utility ofstimulating the AIR to induce regression of a brain tumor.

A 63 year old white male presented with headache and memory loss.Radiographic studies revealed the presence of large stellategliobastoma. The tumor was partially resected surgically, followed bylocal radiation. The subject was placed on a clinical protocolevaluating everolimus and temozolimide.

In mid October 2013, the subject began squalamine capsules with a 100 mgsingle dose. In early December 2013, an MRI revealed that the tumor masshad decreased by about 20%, and a second study in the beginning ofFebruary 2014 revealed continued shrinkage of the residual tumor to 60%of its volume after the surgery/radiation. Squalamine treatmentcontinues.

Example 18

The purpose of this example was to demonstrate the utility ofstimulating the Aminosterol-Induced GI Response to induce repression oflymphoma.

A 92 year old white female with a 2 year history of progressive dementiaand weakness presented with a massive cervical, axillary, and inguinallymphadenopathy, along with malaise, somnolence and pain. Clinicalchemistries revealed mild hypercalcemia, increased polyclonalimmunoglobulins, and no abnormal cells in the blood. An MRI of thecervical area revealed massive adenopathy, with several nodes as largeas walnuts. The skin of her feet was a nonblanching bright pink hue upto the ankle with a scattered brownish purpuric rash. The individual wasprovided end of life care.

At the end of December 2013, the individual was begun on a daily dose ofone 50 mg capsule of squalamine. Previously constipated, bowels began tomove. Rash cleared within 3 days. On day 4 after squalamine initiation,a short course of dexamethsone was begun to treat the hypercalcemia, andstimulate appetite. 20 mg (4 days), 16 mg (2 days), 8 mg, 4 mg (2 days),then 2 mg daily. By 1 week a marked reduction in the size of the nodeswas noted. Mid-January 2014 weakness on the right, involving arm and legwas noted; a diagnosis of a mild stroke was made. Physical examinationat the end of February 2014 revealed no palpable cervical nodes. A flatleft submandibular node was felt of normal size and consistency. Arepeat clinical chemistry study demonstrated complete resolution of thepolyclonal gammopathy and return to normal serum values. Squalaminetreatment continues.

Example 19

The purpose of this example was to demonstrate the utility ofstimulating the Aminosterol-Induced GI Response to induce regression ofa sarcoma and the method by which dosing is arrived at.

A 22 year old man was seen because of the recurrence of an embryonalrhabdomyosarcoma. At age 20 he noticed a mass in his left groin. An MRIrevealed a soft tissue mass; and a biopsy revealed the mass to be aspindle cell embryonal rhabdomyosarcoma. The mass was surgicallyexcised. The patient was then treated with a cocktail ofchemotherapeutic agents. Several months later the mass was seen to recurin the left groin. The mass was again excised. Several months later, atumor was gain noted in the groin, with a large mass present in the leftlung.

The patient was begun on 100 mg capsules of squalamine for several days,without any noticeable GI symptoms. The dose was raised to 200 mg daily,again without any GI response. At 300 mg daily, a clear effect wasnoted, that being mild nausea, 2 hours after dosing, followed by a bowelmovement later in the day. 3 weeks after initiation of dosing the lungtumor was removed surgically and exhibited a massive internal hemorrhagedue to blood vessel damage; dosing continued. 2 months post surgerythere was no evidence of pulmonary tumors nor evidence of recurrence ofany soft tissue tumor in the groin.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodifications and variations of this invention, provided they comewithin the scope of the appended claims and their equivalents.

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What is claimed:
 1. A method of treating a gastrointestinal motilitydisorder in a subject in need thereof, comprising orally administeringto the subject a composition comprising a pharmaceutically acceptablegrade of at least one aminosterol, or a pharmaceutically acceptable saltor derivative thereof, in an amount sufficient to produce a beneficialeffect, wherein: (a) the at least one aminosterol comprises the compoundbelow:

(b) the disorder or condition is selected from the group consisting ofchronic idiopathic constipation, Irritable bowel syndrome,Opioid-induced constipation, and Inflammatory Bowel Disease.
 2. Themethod of claim 1, wherein the disorder or condition is chronicidiopathic constipation.
 3. The method of claim 1, wherein the conditionor disorder is Irritable bowel syndrome.
 4. The method of claim 1,wherein the condition or disorder is Opioid-induced constipation.
 5. Themethod of claim 1, wherein the condition or disorder is InflammatoryBowel Disease.
 6. The method of claim 1, wherein the effective dailydosing amount is about 0.1 to about 20 mg/kg body weight.
 7. The methodof claim 1, wherein the effective dose is established by defining theinitial dose required to induce an Aminosterol-Induced GI Response,which is the initial dose required to stimulate a change in bowelactivity, nausea, secretory diarrhea, or any combination thereof.
 8. Themethod of claim 1, wherein the oral dosage form is a liquid, capsule, ortablet designed to disintegrate in either the stomach, upper smallintestine, or more distal portions of the intestine.
 9. The method ofclaim 1, wherein less than about 10 ng/ml of the administeredaminosterol is detected in the blood stream of the subject about 1 toabout 12 hours following oral administration.
 10. The method of claim 1,wherein the composition further comprises at least one pharmaceuticallyacceptable carrier.
 11. The method of claim 1, wherein the subject ishuman.
 12. The method of claim 1, wherein colonic motility is increasedin the subject following administration of the at least one aminosterol.13. The method of claim 1, wherein an amount sufficient to produce abeneficial effect is an amount sufficient to stimulate the intrinsicprimary afferent neurons (IPANs) in the colon of the subject.
 14. Themethod of claim 1, wherein the at least one aminosterol is administeredin combination with at least one additional active agent to achieveeither an additive or synergistic effect.
 15. The method of claim 14,wherein the additional active agent is administered concomitantly. 16.The method of claim 14, wherein the additional active agent isadministered as an admixture.
 17. The method of claim 14, wherein theadditional active agent is administered separately and simultaneously orconcurrently.
 18. The method of claim 14, wherein the additional activeagent is administered separately and sequentially.